Digital analyte analysis

ABSTRACT

The invention generally relates to droplet based digital PCR and methods for analyzing a target nucleic acid using the same. In certain embodiments, methods of the invention involve forming sample droplets containing, on average, a single target nucleic acid, amplifying the target in the droplets, excluding droplets containing amplicon from the target and amplicon from a variant of the target, and analyzing target amplicons.

REFERENCE TO RELATED APPLICATIONS

This non-provisional application is a continuation of U.S.Non-Provisional application Ser. No. 13/026,120 filed Feb. 11, 2011which claims priority to U.S. Provisional Application No. 61/388,937,filed Oct. 1, 2010, U.S. Provisional Application No. 61/347,158, filedMay 21, 2010, U.S. Provisional Application No. 61/331,490, filed, May 5,2010, and U.S. Provisional Application No. 61/304,163, filed Feb. 12,2010, the contents of which are each incorporated by reference herein intheir entireties.

FIELD OF THE INVENTION

The invention generally relates to droplet based digital PCR and methodsfor analyzing a target nucleic acid using the same.

BACKGROUND

Assays have been developed that rely on analyzing nucleic acid moleculesfrom bodily fluids for the presence of mutations, thus leading to earlydiagnosis of certain diseases such as cancer. In a typical bodily fluidsample however, any abnormal nucleic acids containing mutations ofinterest are often present in small amounts (e.g., less than 1%)relative to a total amount of nucleic acid in the bodily fluid sample.This can result in a failure to detect the small amount of abnormalnucleic acid due to stochastic sampling bias.

The advent of PCR and real-time PCR methodologies has greatly improvedthe analysis of nucleic acids from both throughput and quantitativeperspectives. While traditional PCR techniques typically rely onend-point, and sometimes semi-quantitative, analysis of amplified DNAtargets via agarose gel electrophoresis, real-time PCR (or qPCR) methodsare geared toward accurately quantifying exponential amplification asthe reaction progresses. qPCR reactions are monitored either using avariety of highly sequence specific fluorescent probe technologies, orby using non-specific DNA intercalating fluorogenic dyes.

As the need for higher throughput in analyzing multiple targets inparallel continues to escalate in the fields of genomics and genetics,and as the need for more efficient use of sample grows in medicallyrelated fields such as diagnostics, the ability to perform and quantifymultiple amplifications simultaneously within the same reaction volume(multiplexing) is paramount for both PCR and qPCR. While end-point PCRcan support a high level of amplicon multiplexing, such ample capacityfor multiplexing probe-based qPCR reactions remains elusive for a numberof reasons. For example, most commercial real-time thermal cyclers onlysupport up to four differently colored fluorophores for detection as aconsequence of the limited spectral resolution of common fluorophores,translating into a multiplexing capacity of 4×. Additionally, whileoptimization of single target primer/probe reactions is now standardpractice, combining primers and probes for multiple reactions changesthe thermodynamic efficiencies and/or chemical kinetics, necessitatingpotentially extensive troubleshooting and optimization. Very highmultiplexing of greater than 100× has been demonstrated in a “one ofmany” detection format for pathogen identification using “sloppy”molecular beacons and melting points as fingerprints, however theapproach is restricted to applications with a slim likelihood of thepresence of multiple simultaneous targets. A half-multiplexing methodachieved 19× in a two step reaction with general multiplexedpreamplification in the first step, followed by separate single-plexquantitative PCR in the second step. However a general purposesingle-pot solution to qPCR multiplexing does not yet exist.

Digital PCR (dPCR) is an alternative quantitation method in which dilutesamples are divided into many separate reactions. See for example, Brownet al. (U.S. Pat. Nos. 6,143,496 and 6,391,559) and Vogelstein et al.(U.S. Pat. Nos. 6,440,706, 6,753,147, and 7,824,889), the content ofeach of which is incorporated by reference herein in its entirety. Thedistribution from background of target DNA molecules among the reactionsfollows Poisson statistics, and at so called “terminal dilution” thevast majority of reactions contain either one or zero target DNAmolecules for practical intents and purposes. In another case, at socalled “limiting dilution” some reactions contain zero DNA molecules,some reactions contain one molecule, and frequently some other reactionscontain multiple molecules, following the Poisson distribution. It isunderstood that terminal dilution and limiting dilution are usefulconcepts for describing DNA loading in reaction vessels, but they haveno formal mathematical definition, nor are they necessarily mutuallyexclusive. Ideally, at terminal dilution, the number of PCR positivereactions (PCR(+)) equals the number of template molecules originallypresent. At limiting dilution, Poisson statistics are used to uncoverthe underlying amount of DNA. The principle advantage of digitalcompared to qPCR is that it avoids any need to interpret the timedependence of fluorescence intensity—an analog signal—along with themain underlying uncertainty of non-exponential amplification duringearly cycles.

SUMMARY

The invention generally relates to the manipulation of nucleic acid indroplets, and in particular, nucleic acid amplification and detection.In one aspect, the invention provides a droplet that contains a singlenucleic acid template and a plurality of primer pairs specific formultiple target sites on the template. The single nucleic acid templatecan be DNA (e.g., genomic DNA, cDNA, etc.) or RNA. The template isamplified in the droplet for detection; and may preferably be amplifiedusing a plurality of primer pairs as described herein.

The ability to amplify and detect single nucleic acids in dropletsenables digital PCR, detection, counting, and differentiation amongnucleic acids, especially those present in heterogeneous samples. Thus,the invention applies to digital amplification techniques and, inspecific embodiments enables multiplex PCR in droplets. For example,multiplexing primers in droplets enables the simultaneous increase inthe number of PCR droplets while keeping the amount of input DNA thesame or lower and generate the same or greater amplicon yield. Thisresults in an overall increase in the amount of PCR positive dropletsand amplicon yield without the consumption of more DNA. Even though thenumber of PCR primer pairs per droplet is greater than one, there isonly one template molecule per droplet, and thus, in someimplementations, there is only one primer pair per droplet that is beingutilized at one time. As such, the advantages of droplet PCR foreliminating bias from either allele specific PCR or competition betweendifferent amplicons is maintained. However, as described below inrelation to detection of haplotypes, other implementationsadvantageously allow detection of multiple loci on a single templateusing multiple primer pairs, preferably designed to minimize bias.

Microfluidic droplets for multiplex analysis according to the inventioncontain a plurality of probes that hybridize to amplicons produced inthe droplets. Preferably, the droplet contains two or more probes, e.g.,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 60, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 500, or more probes. Certain members of theplurality of probes include a detectable label. Members of the pluralityof probes can each include the same detectable label, or a differentdetectable label. The detectable label is preferably a fluorescentlabel. The plurality of probes can include one or more groups of probesat varying concentrations. The one or more groups of probes can includethe same detectable label which will vary in intensity upon detection,due to the varying probe concentrations. The droplets of the inventioncan further contain one or more reagents for conducting a polymerasechain reaction, such as a DNA or RNA polymerase, and/or dNTPs.

The present invention additionally relates to a method for detecting aplurality of targets in a biological sample using digital PCR inmicrofluidic droplets. The sample may be a human tissue or body fluid.Exemplary body fluids pus, sputum, semen, urine, blood, saliva, andcerebrospinal fluid.

One or more droplets are formed, each containing a single nucleic acidtemplate and a heterogeneous mixture of primer pairs and probes, eachspecific for multiple target sites on the template. For example, a firstfluid (either continuous, or discontinuous as in droplets) containing asingle nucleic acid template (DNA or RNA) is merged with a second fluid(also either continuous, or discontinuous as in droplets) containing aplurality of primer pairs and a plurality of probes, each specific formultiple targets sites on the nucleic acid template to form a dropletcontaining the single nucleic acid template and a heterogeneous mixtureof primer pairs and probes. The second fluid can also contain reagentsfor conducting a PCR reaction, such as a polymerase and dNTPs.

Certain members of the plurality of probes include a detectable label.Members of the plurality of probes can each include the same detectablelabel, or a different detectable label. The detectable label ispreferably a fluorescent label. The plurality of probes can include oneor more groups of probes at varying concentrations. The one or moregroups of probes can include the same detectable label which varies inintensity upon detection, due to the varying probe concentrations.

The first and second fluids can each be in droplet form. Any techniqueknown in the art for forming droplets may be used with methods of theinvention. An exemplary method involves flowing a stream of the samplefluid containing the nucleic acid template such that it intersects twoopposing streams of flowing carrier fluid. The carrier fluid isimmiscible with the sample fluid. Intersection of the sample fluid withthe two opposing streams of flowing carrier fluid results inpartitioning of the sample fluid into individual sample dropletscontaining the first fluid. The carrier fluid may be any fluid that isimmiscible with the sample fluid. An exemplary carrier fluid is oil. Incertain embodiments, the carrier fluid includes a surfactant, such as afluorosurfactant. The same method may be applied to create individualdroplets from the second fluid containing the primer pairs (and, in someimplementations, the amplification reagents). Either the dropletscontaining the first fluid, the droplets containing the second fluid, orboth, may be formed and then stored in a library for later merging,aspects of certain implementations of which are described in U.S. patentapplication Ser. No. 12/504,764, hereby incorporated herein in itsentirety for all purposes. Once formed, droplets containing the firstand second fluids can be merged to form single droplets containing thesingle nucleic acid template and heterogeneous mixture of primer pairsand probes. Merging can be accomplished, for example, in the presence ofan electric field. Moreover, it is not required that both fluids be inthe form of droplets when merging takes places. One exemplary method formerging of fluid portions with droplets is taught, for example, inco-pending U.S. Patent Application No. 61/441,985, filed on even dateherewith.

The nucleic acid template in each of the merged/formed droplets isamplified, e.g., by thermocycling the droplets undertemperatures/conditions sufficient to conduct a PCR reaction. Theresulting amplicons in the droplets can then be analyzed. For example,the presence of absence of the plurality of targets in the one or moredroplets is detected optically, e.g., by the detectable label on theplurality of probes.

The invention further relates to methods for analyzing a target nucleicacid. More particularly, methods of the invention are able to detectpolymerase errors that occur during a PCR reaction and are able toexclude from analysis amplification products that are a result of apolymerase error. Methods of the invention are particularly useful indigital PCR where a polymerase error may result in a partitioned sectionof sample being incorrectly identified as containing a mutant allele,i.e., a false positive. Such false positives greatly impact the validityand precision of digital PCR results. Methods of the invention are ableto uniquely detect multiple targets with the same optical color. Methodsof the invention are particularly useful in digital PCR where it isdesirable to identify multiple different target molecules that may bepresent in the starting test fluid.

Methods of the invention involve forming sample droplets containingtarget nucleic acid. Ideally, methods of the invention comprise formingdroplets for digital PCR. Preferred digital PCR droplets contain onecopy of a nucleic acid to be amplified, although they may containmultiple copies of the same nucleic acid sequence. Any technique knownin the art for forming sample droplets may be used with methods of theinvention. One exemplary method involves flowing a stream of samplefluid including nucleic acids such that it intersects two opposingstreams of flowing carrier fluid. The carrier fluid is immiscible withthe sample fluid. Intersection of the sample fluid with the two opposingstreams of flowing carrier fluid results in partitioning of the samplefluid into individual sample droplets. The carrier fluid may be anyfluid that is immiscible with the sample fluid. An exemplary carrierfluid is oil. In certain embodiments, the carrier fluid includes asurfactant, such as a fluorosurfactant.

The targets are then amplified in the droplets. Any method known in theart may be used to amplify the target nucleic acids either linearly orexponentially. A preferred method is the polymerase chain reaction(PCR). For purposes of the invention, any amplification techniquecommonly known in the art may be implemented such as rolling circleamplification, isothermal amplification, or any combination ofamplification methods using loci specific primers, nested-primers, orrandom primers (such primers, and/or primers used for PCR, are includedin the term “amplification reagents”). Once amplified, dropletscontaining amplicon from the target and amplicon from a variant of thetarget are excluded. One method to exclude droplets that contain aheterogeneous population of amplicons from droplets that contain ahomogeneous population of amplicons includes hybridizingdetectably-labeled probes to the amplicons, flowing the droplets througha microfluidic channel, and excluding those droplets in which bothamplicon from the target and amplicon from a variant of the target aredetected.

Once droplets containing a heterogeneous population of amplicons areexcluded, droplets that contain a homogeneous population of ampliconsare analyzed. Any analytical technique known in the art may be used. Incertain embodiments, analyzing the droplets involves determining anumber of droplets that contain only wild-type target, and determining anumber of droplets that contain only a variant of the target. Generally,the presence of droplets containing only the variant is indicative of adisease, such as cancer. The variant may be an allelic variant. Anexemplary allelic variant is a single nucleotide polymorphism. Thevariant may also be a specific haplotype. Haplotypes refer to thepresence of two or more variants on the same nucleic acid strand.Haplotypes can be more informative or predictive than genotypes whenused to determine such things as the presence or severity of disease,response to drug therapy or drug resistance of bacterial or viralinfections. Because each droplet contains only one template strand it isan ideal vessel for the determination of haplotypes. The detection oftwo or more variants in a single droplet that contains a single intactnucleic acid strand identifies the haplotype of the variants on thatstrand. The presence of two or more markers in the same droplet can beidentified by such methods as the presence of dyes of multiple colors orthe increase in the intensity of a single dye or a combination of both.Any method that allows the identification of multiple variants in asingle droplet enables the determination of a samples haplotype.

In accordance with some implementations of the invention, a method isprovided for analyzing a target nucleic acid that includescompartmentalizing a first fluid into portions, each portion containinga single target nucleic acid; amplifying the target in the portions;excluding portions containing amplicon from the target and amplicon froma variant of the target; and analyzing target amplicons.

In other aspects, the invention generally provides methods for detectinga recurrence of a cancer in a patient. Those methods may involve formingsample droplets containing a single target nucleic acid derived from apatient sample, flowing the sample droplets through a channel,amplifying the target in the droplets, detecting amplified target in thedroplets, excluding droplets including a heterogeneous population ofamplicons, and analyzing non-excluded droplets to determine the presenceof mutant alleles indicative of recurrence. In certain embodiments, theanalyzing step includes capturing amplicon obtained from the dropletsusing labeled capture probes. The sample may be a human tissue or bodyfluid. Exemplary body fluids are pus, sputum, semen, urine, blood,saliva, stool, and cerebrospinal fluid. In other aspects of theinvention generally provide a method for forensic identification of lowlevels of target nucleic acid in an environment having multiple othersources of nucleic acid. Such methods may also be practiced using fluidscompartmentalized in containers other than or in addition to droplets.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a droplet formation device.

FIG. 2 depicts a portion of the droplet formation device of FIG. 1.

FIGS. 3 a-3 c depict an exemplary microfluidic system for dropletgeneration and readout. FIG. 3 a depicts the droplet generation chip;FIG. 3 b depicts the droplet spacing for readout; and FIG. 3 c depicts acartoon of droplet readout by fluorescence.

FIG. 4 panels 4 a-4-c depicts the serial dilution of template DNAquantified by dPCR. FIG. 4 a shows droplet fluorescence during readoutfor the most concentrated sample. Each discrete burst of fluorescencecorresponded to an individual droplet. Two different groups of dropletswere evident: PCR(+) droplets peaking at ˜0.8 V and PCR(−) droplets at˜0.1 V; FIG. 4 b shows a histogram of the peak fluorescence intensitiesof droplets from the complete data trace in (a). PCR(+) and PCR(−)droplets appeared as two very distinct populations centered at 0.78 and0.10 V, respectively; FIG. 4 c shows the serial dilution of templateDNA. Open circles: measured occupancies; solid line: the best fit to Eqn2 (A=0.15, f=4.8, R² −0.9999).

FIG. 5A is a schematic representation of a droplet having 5 sets ofprimers for PCR amplification of a template sequence and 5 probes, eachlabeled with a fluorescent dye, that binds specifically to the amplifiedsequences; FIG. 5B is a time trace of fluorescence intensity detectedfrom droplets after PCR amplification; FIG. 5C is a scatter plot showingclusters representing droplets that contain specific amplified sequences(TERT, RNaseP, E1a, SMN1 and SMN2).

FIG. 6A is a schematic representation of a droplet having 5 sets ofprimers for PCR amplification of a template sequence and 5 probes, eachlabeled with a fluorescent dye, that binds specifically to the amplifiedsequences; FIG. 6B is a scatter plot showing clusters representingdroplets that contain specific amplified sequences (TERT, 815A, RNaseP,E1a, and 815G); FIG. 6C is a table showing the copy number of specificsequences shown in FIG. 6B.

FIGS. 7A-7E are schematics depicting one-color detection of a geneticsequence with a microfluidic device.

FIGS. 8A-8D are schematics depicting two-color detection of two geneticsequences with a microfluidic device.

FIGS. 9A-9D are schematics depicting two-color detection of threegenetic sequences with a microfluidic device.

FIG. 10 shows two dot plots depicting clusters of genetic sequencesdetected through fluorescence intensity. Left panel is a dot plotshowing four clusters. Block for SMN1 sequence was present. Top left:microdroplets containing the reference sequence (SMARCC1); bottom left:microdroplets not containing any sequence; bottom middle: microdropletscontaining sequence for SMN1; and bottom right: microdroplets containingsequence for SMN2. Right panel is a dot plot showing four clusters. Noblock for SMN1 sequence was present. Top left: microdroplets containingthe reference sequence (SMARCC1); bottom left: microdroplets notcontaining any sequence; bottom middle: microdroplets containingsequence for SMN1; and bottom right: microdroplets containing sequencefor SMN2. The shift of the bottom middle cluster in right panel ascompared to left panel confirms that fluorescence intensity provides avery sensitive measurement for the presence of a sequence.

FIGS. 11 a-11 b depicts histograms of a duplex gene copy number assayusing only one type of fluorophore by digital PCR; FIG. 11 a depicts ahistogram of droplet peak fluorescence intensities; FIG. 11 b shows acomparison of gene copy numbers measured by monochromatic dPCR.

FIGS. 12A-12C are schematics for tuning the intensity of a detectablelabel to a particular target with a microfluidic device.

FIG. 13 is a line graph depicting the linear dependence of dropletfluorescence intensity on probe concentration (Line, best linear fit(y=−0.092x+0.082, R²=0.995).

FIGS. 14A-14B depict a 5-plex dPCR assay for spinal muscular atrophywith only two fluorophores. FIG. 14 a is a 2D histogram of dropletfluorescence intensities, shown as a heat map, for the 5-plex assayagainst the synthetic model chromosome for validation. The six wellresolved droplet populations corresponded to the five individual assaysplus the empty droplets; FIG. 14 b shows the results of the SMA pilotstudy.

FIG. 15 depicts a 9-plex dPCR assay for spinal muscular atrophy withonly two fluorophores, showing the process of optimizing dropletintensities. FIG. 15 shows 2-D histograms of droplet fluorescenceintensity, shown as heat maps with hotter colors representing higherdroplet counts, for the 9-plex assay against the synthetic modelchromosome (left panel=Before optimization; right panel=afteroptimization).

FIG. 16 depicts an optical schematic for combining optical labels withmultiplexing.

FIG. 17 depicts a dPCR assay combining multiplexing with optical labelsusing co-flow microfluidics. The contributions from all droplets areshown, that is, from three different triplex assays. (Both panels) 2-Dhistograms shown as heat maps with hotter colors representing higherdroplet counts. (Left panel) histogram of optical labels, i.e.fluorescence intensities of droplets measured at wavelengths for the twofluorophores comprising the optical labels. (Right panel) assayhistogram, i.e. fluorescence intensities of droplets measured atwavelengths suitable for FAM detection (x-axis), and VIC detection(y-axis). Both histograms were compensated for spectral overlap bystandard techniques.

FIGS. 18A-18C show single assay selections using optical labels.Selections were taken from all of the droplets from FIG. 17. Each of thethree different selections in panels A-C were for optical labelsencoding the same assay (TERT, SMN1, and SMN2). Histograms are asdescribed in FIG. 17. (Left histograms, optical labels) Superimposedlines demark the bounding box for selecting a single optical label.(Right histograms, assay) Only droplets containing the selected opticallabel are displayed.

FIGS. 19A-19C show single assay selections using optical labels.Selections were taken from all of the droplets from FIG. 17. Each of thethree different selections in panels A-C was for optical labels encodingthe same assay (TERT, c.5C from SMN1, and BCKDHA). Histograms are asdescribed in FIG. 17. (Left histograms, optical labels) Superimposedlines demark the bounding box for selecting a single optical label.(Right histograms, assay) Only droplets containing the selected opticallabel are displayed.

FIGS. 20A-20C show single assay selections using optical labels.Selections were taken from all of the droplets from FIG. 17. Each of thethree different selections in panels A-C was for optical labels encodingthe same assay (TERT, c.88G from SMN1, and RNaseP). Histograms are asdescribed in FIG. 17. (Left histograms, optical labels) Superimposedlines demark the bounding box for selecting a single optical label.(Right histograms, assay) Only droplets containing the selected opticallabel are displayed.

FIGS. 21A-21J depict a dPCR assay combining multiplexing with opticallabels using droplet merging.

FIG. 22 is a schematic showing haplotype detection in droplets.

DETAILED DESCRIPTION

The invention provides materials and methods for analysis ofbiomolecules. In one aspect, the invention provides for digital analysisin droplets, such as microfluidic droplets. The invention allows digitalPCR to be conducted and provides for significantly reduced or eliminatederrors.

Ideally, the sensitivity of digital PCR is limited only by the number ofindependent amplifications that can be analyzed, which has motivated thedevelopment of several ultra-high throughput miniaturized methodsallowing millions of single molecule PCR reactions to be performed inparallel (discussed in detail elsewhere). In a preferred embodiment ofthe invention, digital PCR is performed in aqueous droplets separated byoil using a microfluidics system. In another preferred embodiment, theoil is a fluorinated oil such as the Fluorinert oils (3M). In a stillmore preferred embodiment the fluorinated oil contains a surfactant,such as PFPE-PEG-PFPE triblock copolymer, to stabilize the dropletsagainst coalescence during the amplification step or at any point wherethey contact each other. Microfluidic approaches allow the rapidgeneration of large numbers (e.g. 106 or greater) of very uniformlysized droplets that function as picoliter volume reaction vessels (seereviews of droplet-based microfluidics). But as will be described, theinvention is not limited to dPCR performed in water-in-oil emulsions,but rather is general to all methods of reaction compartmentalizationfor dPCR. In the description that follows, the invention is described interms of the use of droplets for compartmentalization, but it isunderstood that this choice of description is not limiting for theinvention, and that all of the methods of the invention are compatiblewith all other methods of reaction compartmentalization for dPCR.

Methods of the invention involve novel strategies for performingmultiple different amplification reactions on the same samplesimultaneously to quantify the abundance of multiple different DNAtargets, commonly known to those familiar with the art as“multiplexing”. Methods of the invention for multiplexing dPCR assayspromise greater plexity—the number of simultaneous reactions—thanpossible with existing qPCR or dPCR techniques. It is based on thesingular nature of amplifications at terminal or limiting dilution thatarises because most often only a single target allele is ever present inany one droplet even when multiple primers/probes targeting differentalleles are present. This alleviates the complications that otherwiseplague simultaneous competing reactions, such as varying arrival timeinto the exponential stage and unintended interactions between primers.

In one aspect, the invention provides materials and methods forimproving amplicon yield while maintaining the sensitivity andspecificity in droplet based digital PCR. More specifically, theinvention provides droplets containing a single nucleic acid templateand multiplexed PCR primers and methods for detecting a plurality oftargets in a biological sample by forming such droplets and amplifyingthe nucleic acid templates using droplet based digital PCR.

Reactions within microfluidic droplets yield very uniform fluorescenceintensity at the end point, and ultimately the intensity depends on theefficiency of probe hydrolysis. Thus, in another aspect of the methodsof the invention, different reactions with different efficiencies can bediscriminated on the basis of end point fluorescence intensity aloneeven if they have the same color. Furthermore, in another method of theinvention, the efficiencies can be tuned simply by adjusting the probeconcentration, resulting in an easy-to-use and general purpose methodfor multiplexing. In one demonstration of the invention, a 5-plexTaqMan® dPCR assay worked “right out of the box”, in contrast to lengthyoptimizations that typify qPCR multiplexing to this degree. In anotheraspect of the invention, adding multiple colors increases the number ofpossible reactions geometrically, rather than linearly as with qPCR,because individual reactions can be labeled with multiple fluorophores.As an example, two fluorophores (VIC and FAM) were used to distinguishfive different reactions in one implementation of the invention.

Methods of the invention are able to detect polymerase errors that occurduring an amplification reaction and are able to exclude from analysisthose products that are a result of polymerase errors. In essence,methods of the invention increase the sensitivity of digital PCR byidentifying amplification products that are false positives, andexcluding those products from analysis.

Methods of the invention involve forming sample droplets containing asingle target nucleic acid, amplifying the target in the droplets,excluding droplets containing amplicon from the target and amplicon froma variant of the target, and analyzing target amplicons.

Nucleic Acid Target Molecules

Nucleic acid molecules include deoxyribonucleic acid (DNA) and/orribonucleic acid (RNA). Nucleic acid molecules can be synthetic orderived from naturally occurring sources. In one embodiment, nucleicacid molecules are isolated from a biological sample containing avariety of other components, such as proteins, lipids and non-templatenucleic acids. Nucleic acid template molecules can be obtained from anycellular material, obtained from an animal, plant, bacterium, fungus, orany other cellular organism. In certain embodiments, the nucleic acidmolecules are obtained from a single cell. Biological samples for use inthe present invention include viral particles or preparations. Nucleicacid molecules can be obtained directly from an organism or from abiological sample obtained from an organism, e.g., from blood, urine,cerebrospinal fluid, seminal fluid, saliva, sputum, stool and tissue.Any tissue or body fluid specimen may be used as a source for nucleicacid for use in the invention. Nucleic acid molecules can also beisolated from cultured cells, such as a primary cell culture or a cellline. The cells or tissues from which template nucleic acids areobtained can be infected with a virus or other intracellular pathogen. Asample can also be total RNA extracted from a biological specimen, acDNA library, viral, or genomic DNA. In certain embodiments, the nucleicacid molecules are bound as to other target molecules such as proteins,enzymes, substrates, antibodies, binding agents, beads, small molecules,peptides, or any other molecule and serve as a surrogate for quantifyingand/or detecting the target molecule.

Generally, nucleic acid can be extracted from a biological sample by avariety of techniques such as those described by Maniatis, et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., pp.280-281 (1982). Nucleic acid molecules may be single-stranded,double-stranded, or double-stranded with single-stranded regions (forexample, stem- and loop-structures).

Droplet Formation

Methods of the invention involve forming sample droplets where somedroplets contain zero target nucleic acid molecules, some dropletscontain one target nucleic acid molecule, and some droplets may or maynot contain multiple nucleic acid molecules (corresponding to limitingor terminal dilution, respectively, as defined above). In the preferredembodiment, the distribution of molecules within droplets obeys thePoisson distribution. However, methods for non-Poisson loading ofdroplets are known to those familiar with the art, and include but arenot limited to active sorting of droplets, such as by laser-inducedfluorescence, or by passive one-to-one loading. The description thatfollows assumes Poisson loading of droplets, but such description is notintended to exclude non-Poisson loading, as the invention is compatiblewith all distributions of DNA loading that conform to limiting orterminal dilution.

The droplets are aqueous droplets that are surrounded by an immisciblecarrier fluid. Methods of forming such droplets are shown for example inLink et al. (U.S. patent application numbers 2008/0014589, 2008/0003142,and 2010/0137163), Stone et al. (U.S. Pat. No. 7,708,949 and U.S. patentapplication number 2010/0172803), Anderson et al. (U.S. Pat. No.7,041,481 and which reissued as U.S. Pat. No. RE 41,780) and Europeanpublication number EP2047910 to Raindance Technologies Inc. The contentof each of which is incorporated by reference herein in its entirety.

FIG. 1 shows an exemplary embodiment of a device 100 for dropletformation. Device 100 includes an inlet channel 101, and outlet channel102, and two carrier fluid channels 103 and 104. Channels 101, 102, 103,and 104 meet at a junction 105. Inlet channel 101 flows sample fluid tothe junction 105. Carrier fluid channels 103 and 104 flow a carrierfluid that is immiscible with the sample fluid to the junction 105.Inlet channel 101 narrows at its distal portion wherein it connects tojunction 105 (See FIG. 2). Inlet channel 101 is oriented to beperpendicular to carrier fluid channels 103 and 104. Droplets are formedas sample fluid flows from inlet channel 101 to junction 105, where thesample fluid interacts with flowing carrier fluid provided to thejunction 105 by carrier fluid channels 103 and 104. Outlet channel 102receives the droplets of sample fluid surrounded by carrier fluid.

The sample fluid is typically an aqueous buffer solution, such asultrapure water (e.g., 18 mega-ohm resistivity, obtained, for example bycolumn chromatography), 10 mM Tris HCl and 1 mM EDTA (TE) buffer,phosphate buffer saline (PBS) or acetate buffer. Any liquid or bufferthat is physiologically compatible with nucleic acid molecules can beused. The carrier fluid is one that is immiscible with the sample fluid.The carrier fluid can be a non-polar solvent, decane (e.g., tetradecaneor hexadecane), fluorocarbon oil, silicone oil or another oil (forexample, mineral oil).

In certain embodiments, the carrier fluid contains one or moreadditives, such as agents which increase, reduce, or otherwise createnon-Newtonian surface tensions (surfactants) and/or stabilize dropletsagainst spontaneous coalescence on contact. Surfactants can includeTween, Span, fluorosurfactants, and other agents that are soluble in oilrelative to water. In some applications, performance is improved byadding a second surfactant, or other agent, such as a polymer or otheradditive, to the sample fluid. Surfactants can aid in controlling oroptimizing droplet size, flow and uniformity, for example by reducingthe shear force needed to extrude or inject droplets into anintersecting channel. This can affect droplet volume and periodicity, orthe rate or frequency at which droplets break off into an intersectingchannel. Furthermore, the surfactant can serve to stabilize aqueousemulsions in fluorinated oils from coalescing.

In certain embodiments, the droplets may be coated with a surfactant ora mixture of surfactants. Preferred surfactants that may be added to thecarrier fluid include, but are not limited to, surfactants such assorbitan-based carboxylic acid esters (e.g., the “Span” surfactants,Fluka Chemika), including sorbitan monolaurate (Span 20), sorbitanmonopalmitate (Span 40), sorbitan monostearate (Span 60) and sorbitanmonooleate (Span 80), and perfluorinated polyethers (e.g., DuPont Krytox157 FSL, FSM, and/or FSH). Other non-limiting examples of non-ionicsurfactants which may be used include polyoxyethylenated alkylphenols(for example, nonyl-, p-dodecyl-, and dinonylphenols),polyoxyethylenated straight chain alcohols, polyoxyethylenatedpolyoxypropylene glycols, polyoxyethylenated mercaptans, long chaincarboxylic acid esters (for example, glyceryl and polyglycerl esters ofnatural fatty acids, propylene glycol, sorbitol, polyoxyethylenatedsorbitol esters, polyoxyethylene glycol esters, etc.) and alkanolamines(e.g., diethanolamine-fatty acid condensates and isopropanolamine-fattyacid condensates).

In certain embodiments, the carrier fluid may be caused to flow throughthe outlet channel so that the surfactant in the carrier fluid coats thechannel walls. In one embodiment, the fluorosurfactant can be preparedby reacting the perflourinated polyether DuPont Krytox 157 FSL, FSM, orFSH with aqueous ammonium hydroxide in a volatile fluorinated solvent.The solvent and residual water and ammonia can be removed with a rotaryevaporator. The surfactant can then be dissolved (e.g., 2.5 wt %) in afluorinated oil (e.g., Flourinert (3M)), which then serves as thecarrier fluid.

One approach to merging sample fluids, using a device called a lambdainjector, involves forming a droplet, and contacting the droplet with afluid stream, in which a portion of the fluid stream integrates with thedroplet to form a mixed droplet. In this approach, only one phase needsto reach a merge area in a form of a droplet. Further description ofsuch method is shown in the co-owned and co-pending U.S. patentapplication to Yurkovetsky, et al. (U.S. patent application Ser. No.61/441,985), the content of which is incorporated y reference herein inits entirety.

According to a method for operating the lambda injector, a droplet isformed as described above. After formation of the sample droplet fromthe first sample fluid, the droplet is contacted with a flow of a secondsample fluid stream. Contact between the droplet and the fluid streamresults in a portion of the fluid stream integrating with the droplet toform a mixed droplet.

The droplets of the first sample fluid flow through a first channelseparated from each other by immiscible carrier fluid and suspended inthe immiscible carrier fluid. The droplets are delivered to the mergearea, i.e., junction of the first channel with the second channel, by apressure-driven flow generated by a positive displacement pump. Whiledroplet arrives at the merge area, a bolus of a second sample fluid isprotruding from an opening of the second channel into the first channel.Preferably, the channels are oriented perpendicular to each other.However, any angle that results in an intersection of the channels maybe used.

The bolus of the second sample fluid stream continues to increase insize due to pumping action of a positive displacement pump connected tochannel, which outputs a steady stream of the second sample fluid intothe merge area. The flowing droplet containing the first sample fluideventually contacts the bolus of the second sample fluid that isprotruding into the first channel. Contact between the two sample fluidsresults in a portion of the second sample fluid being segmented from thesecond sample fluid stream and joining with the first sample fluiddroplet to form a mixed droplet. In certain embodiments, each incomingdroplet of first sample fluid is merged with the same amount of secondsample fluid.

In certain embodiments, an electric charge is applied to the first andsecond sample fluids. Description of applying electric charge to samplefluids is provided in Link et al. (U.S. patent application number2007/0003442) and European Patent Number EP2004316 to RaindanceTechnologies Inc, the content of each of which is incorporated byreference herein in its entirety. Electric charge may be created in thefirst and second sample fluids within the carrier fluid using anysuitable technique, for example, by placing the first and second samplefluids within an electric field (which may be AC, DC, etc.), and/orcausing a reaction to occur that causes the first and second samplefluids to have an electric charge, for example, a chemical reaction, anionic reaction, a photocatalyzed reaction, etc.

The electric field, in some embodiments, is generated from an electricfield generator, i.e., a device or system able to create an electricfield that can be applied to the fluid. The electric field generator mayproduce an AC field (i.e., one that varies periodically with respect totime, for example, sinusoidally, sawtooth, square, etc.), a DC field(i.e., one that is constant with respect to time), a pulsed field, etc.The electric field generator may be constructed and arranged to createan electric field within a fluid contained within a channel or amicrofluidic channel. The electric field generator may be integral to orseparate from the fluidic system containing the channel or microfluidicchannel, according to some embodiments.

Techniques for producing a suitable electric field (which may be AC, DC,etc.) are known to those of ordinary skill in the art. For example, inone embodiment, an electric field is produced by applying voltage acrossa pair of electrodes, which may be positioned on or embedded within thefluidic system (for example, within a substrate defining the channel ormicrofluidic channel), and/or positioned proximate the fluid such thatat least a portion of the electric field interacts with the fluid. Theelectrodes can be fashioned from any suitable electrode material ormaterials known to those of ordinary skill in the art, including, butnot limited to, silver, gold, copper, carbon, platinum, copper,tungsten, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as wellas combinations thereof. In some cases, transparent or substantiallytransparent electrodes can be used.

The electric field facilitates rupture of the interface separating thesecond sample fluid and the droplet. Rupturing the interface facilitatesmerging of bolus of the second sample fluid and the first sample fluiddroplet. The forming mixed droplet continues to increase in size untilit a portion of the second sample fluid breaks free or segments from thesecond sample fluid stream prior to arrival and merging of the nextdroplet containing the first sample fluid. The segmenting of the portionof the second sample fluid from the second sample fluid stream occurs assoon as the shear force exerted on the forming mixed droplet by theimmiscible carrier fluid overcomes the surface tension whose action isto keep the segmenting portion of the second sample fluid connected withthe second sample fluid stream. The now fully formed mixed dropletcontinues to flow through the first channel.

In other embodiments, the rupture of the interface can be spontaneous,or the rupture can be facilitated by surface chemistry. The invention isnot limited in regard to the method of rupture at the interface, asrupture can be brought about by any means.

In the context of PCR, in a preferred embodiment, the first sample fluidcontains nucleic acid templates. Droplets of the first sample fluid areformed as described above. Those droplets will include the nucleic acidtemplates. In certain embodiments, the droplets will include only asingle nucleic acid template, and thus digital PCR can be conducted. Thesecond sample fluid contains reagents for the PCR reaction. Suchreagents generally include Taq polymerase, deoxynucleotides of type A,C, G and T, magnesium chloride, and forward and reverse primers, allsuspended within an aqueous buffer. The second fluid also includesdetectably labeled probes for detection of the amplified target nucleicacid, the details of which are discussed below. A droplet containing thenucleic acid is then caused to merge with the PCR reagents in the secondfluid as described above, producing a droplet that includes Taqpolymerase, deoxynucleotides of type A, C, G and T, magnesium chloride,forward and reverse primers, detectably labeled probes, and the targetnucleic acid. In another embodiment, the first fluid can contain thetemplate DNA and PCR master mix (defined below), and the second fluidcan contain the forward and reverse primers and the probe. The inventionis not restricted in any way regarding the constituency of the first andsecond fluidics for PCR or digital PCR. For example, in someembodiments, the template DNA is contained in the second fluid insidedroplets.

Target Amplification

Methods of the invention further involve amplifying the target nucleicacid in each droplet. Amplification refers to production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction or other technologies well known in the art(e.g., Dieffenbach and Dveksler, PCR Primer, a Laboratory Manual, ColdSpring Harbor Press, Plainview, N.Y. [1995]). The amplification reactionmay be any amplification reaction known in the art that amplifiesnucleic acid molecules, such as polymerase chain reaction, nestedpolymerase chain reaction, ligase chain reaction (Barany F. (1991) PNAS88:189-193; Barany F. (1991) PCR Methods and Applications 1:5-16),ligase detection reaction (Barany F. (1991) PNAS 88:189-193), stranddisplacement amplification, transcription based amplification system,nucleic acid sequence-based amplification, rolling circle amplification,and hyper-branched rolling circle amplification.

In certain embodiments, the amplification reaction is the polymerasechain reaction. Polymerase chain reaction (PCR) refers to methods by K.B. Mullis (U.S. Pat. Nos. 4,683,195 and 4,683,202, hereby incorporatedby reference) for increasing concentration of a segment of a targetsequence in a mixture of genomic DNA without cloning or purification.The process for amplifying the target sequence includes introducing anexcess of oligonucleotide primers to a DNA mixture containing a desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The primers are complementary to theirrespective strands of the double stranded target sequence.

To effect amplification, primers are annealed to their complementarysequence within the target molecule. Following annealing, the primersare extended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one cycle; there can be numerous cycles) to obtaina high concentration of an amplified segment of a desired targetsequence. The length of the amplified segment of the desired targetsequence is determined by relative positions of the primers with respectto each other and by cycling parameters, and therefore, this length is acontrollable parameter.

Methods for performing PCR in droplets are shown for example in Link etal. (U.S. patent application numbers 2008/0014589, 2008/0003142, and2010/0137163), Anderson et al. (U.S. Pat. No. 7,041,481 and whichreissued as U.S. Pat. No. RE 41,780) and European publication numberEP2047910 to Raindance Technologies Inc. The content of each of which isincorporated by reference herein in its entirety.

The sample droplet may be pre-mixed with a primer or primers, or theprimer or primers may be added to the droplet. In some embodiments,droplets created by segmenting the starting sample are merged with asecond set of droplets including one or more primers for the targetnucleic acid in order to produce final droplets. The merging of dropletscan be accomplished using, for example, one or more droplet mergingtechniques described for example in Link et al. (U.S. patent applicationnumbers 2008/0014589, 2008/0003142, and 2010/0137163) and Europeanpublication number EP2047910 to Raindance Technologies Inc.

In embodiments involving merging of droplets, two droplet formationmodules are used. In one embodiment, a first droplet formation moduleproduces the sample droplets consistent with limiting or terminaldilution of target nucleic acid. A second droplet formation orreinjection module inserts droplets that contain reagents for a PCRreaction. Such droplets generally include the “PCR master mix” (known tothose in the art as a mixture containing at least Taq polymerase,deoxynucleotides of type A, C, G and T, and magnesium chloride) andforward and reverse primers (known to those in the art collectively as“primers”), all suspended within an aqueous buffer. The second dropletalso includes detectably labeled probes for detection of the amplifiedtarget nucleic acid, the details of which are discussed below. Differentarrangements of reagents between the two droplet types is envisioned.For example, in another embodiment, the template droplets also containthe PCR master mix, but the primers and probes remain in the seconddroplets. Any arrangement of reagents and template DNA can be usedaccording to the invention.

Primers can be prepared by a variety of methods including but notlimited to cloning of appropriate sequences and direct chemicalsynthesis using methods well known in the art (Narang et al., MethodsEnzymol., 68:90 (1979); Brown et al., Methods Enzymol., 68:109 (1979)).Primers can also be obtained from commercial sources such as OperonTechnologies, Amersham Pharmacia Biotech, Sigma, and Life Technologies.The primers can have an identical melting temperature. The lengths ofthe primers can be extended or shortened at the 5′ end or the 3′ end toproduce primers with desired melting temperatures. Also, the annealingposition of each primer pair can be designed such that the sequence and,length of the primer pairs yield the desired melting temperature. Thesimplest equation for determining the melting temperature of primerssmaller than 25 base pairs is the Wallace Rule (Td=2(A+T)+4(G+C)).Another method for determining the melting temperature of primers is thenearest neighbor method Computer programs can also be used to designprimers, including but not limited to Array Designer Software (ArrayitInc.), Oligonucleotide Probe Sequence Design Software for GeneticAnalysis (Olympus Optical Co.), NetPrimer, and DNAsis from HitachiSoftware Engineering. The TM (melting or annealing temperature) of eachprimer is calculated using software programs such as Oligo Design,available from Invitrogen Corp.

In one embodiment, the droplet formation modules are arranged andcontrolled to produce an interdigitation of sample droplets and PCRreagent droplets flowing through a channel. Such an arrangement isdescribed for example in Link et al. (U.S. patent application numbers2008/0014589, 2008/0003142, and 2010/0137163) and European publicationnumber EP2047910 to Raindance Technologies Inc.

A sample droplet is then caused to merge with a PCR reagent droplet,producing a droplet that includes the PCR master mix, primers,detectably labeled probes, and the target nucleic acid. Droplets may bemerged for example by: producing dielectrophoretic forces on thedroplets using electric field gradients and then controlling the forcesto cause the droplets to merge; producing droplets of different sizesthat thus travel at different velocities, which causes the droplets tomerge; and producing droplets having different viscosities that thustravel at different velocities, which causes the droplets to merge witheach other. Each of those techniques is further described in Link et al.(U.S. patent application numbers 2008/0014589, 2008/0003142, and2010/0137163) and European publication number EP2047910 to RaindanceTechnologies Inc. Further description of producing and controllingdielectrophoretic forces on droplets to cause the droplets to merge isdescribed in Link et al. (U.S. patent application number 2007/0003442)and European Patent Number EP2004316 to Raindance Technologies Inc.

In another embodiment, called simple droplet generation, a singledroplet formation module, or a plurality of droplet formation modulesare arranged to produce droplets from a mixture already containing thetemplate DNA, the PCR master mix, primers, and detectably labeledprobes. In yet another embodiment, called co-flow, upstream from asingle droplet formation module two channels intersect allowing two flowstreams to converge. One flow stream contains one set of reagents andthe template DNA, and the other contains the remaining reagents. In thepreferred embodiment for co-flow, the template DNA and the PCR mastermix are in one flow stream, and the primers and probes are in the other.However, the invention is not limited in regard to the constituency ofeither flow stream. For example, in another embodiment, one flow streamcontains just the template DNA, and the other contains the PCR mastermix, the primers, and the probes. On convergence of the flow streams ina fluidic intersection, the flow streams may or may not mix before thedroplet generation nozzle. In either embodiment, some amount of fluidfrom the first stream, and some amount of fluid from the second streamare encapsulated within a single droplet. Following encapsulation,complete mixing occurs.

Once final droplets have been produced by any of the droplet formingembodiments above, or by any other embodiments, the droplets are thermalcycled, resulting in amplification of the target nucleic acid in eachdroplet. In certain embodiments, the droplets are collected off-chip asan emulsion in a PCR thermal cycling tube and then thermally cycled in aconventional thermal cycler. Temperature profiles for thermal cyclingcan be adjusted and optimized as with any conventional DNA amplificationby PCR.

In certain embodiments, the droplets are flowed through a channel in aserpentine path between heating and cooling lines to amplify the nucleicacid in the droplet. The width and depth of the channel may be adjustedto set the residence time at each temperature, which can be controlledto anywhere between less than a second and minutes.

In certain embodiments, the three temperature zones are used for theamplification reaction. The three temperature zones are controlled toresult in denaturation of double stranded nucleic acid (high temperaturezone), annealing of primers (low temperature zones), and amplificationof single stranded nucleic acid to produce double stranded nucleic acids(intermediate temperature zones). The temperatures within these zonesfall within ranges well known in the art for conducting PCR reactions.See for example, Sambrook et al. (Molecular Cloning, A LaboratoryManual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 2001).

In certain embodiments, the three temperature zones are controlled tohave temperatures as follows: 95° C. (TH), 55° C. (TL), 72° C. (TM). Theprepared sample droplets flow through the channel at a controlled rate.The sample droplets first pass the initial denaturation zone (TH) beforethermal cycling. The initial preheat is an extended zone to ensure thatnucleic acids within the sample droplet have denatured successfullybefore thermal cycling. The requirement for a preheat zone and thelength of denaturation time required is dependent on the chemistry beingused in the reaction. The samples pass into the high temperature zone,of approximately 95° C., where the sample is first separated into singlestranded DNA in a process called denaturation. The sample then flows tothe low temperature, of approximately 55° C., where the hybridizationprocess takes place, during which the primers anneal to thecomplementary sequences of the sample. Finally, as the sample flowsthrough the third medium temperature, of approximately 72° C., thepolymerase process occurs when the primers are extended along the singlestrand of DNA with a thermostable enzyme. Methods for controlling thetemperature in each zone may include but are not limited to electricalresistance, peltier junction, microwave radiation, and illumination withinfrared radiation.

The nucleic acids undergo the same thermal cycling and chemical reactionas the droplets passes through each thermal cycle as they flow throughthe channel. The total number of cycles in the device is easily alteredby an extension of thermal zones or by the creation of a continuous loopstructure. The sample undergoes the same thermal cycling and chemicalreaction as it passes through N amplification cycles of the completethermal device.

In other embodiments, the temperature zones are controlled to achievetwo individual temperature zones for a PCR reaction. In certainembodiments, the two temperature zones are controlled to havetemperatures as follows: 95° C. (TH) and 60° C. (TL). The sample dropletoptionally flows through an initial preheat zone before entering thermalcycling. The preheat zone may be important for some chemistry foractivation and also to ensure that double stranded nucleic acid in thedroplets are fully denatured before the thermal cycling reaction begins.In an exemplary embodiment, the preheat dwell length results inapproximately 10 minutes preheat of the droplets at the highertemperature.

The sample droplet continues into the high temperature zone, ofapproximately 95° C., where the sample is first separated into singlestranded DNA in a process called denaturation. The sample then flowsthrough the device to the low temperature zone, of approximately 60° C.,where the hybridization process takes place, during which the primersanneal to the complementary sequences of the sample. Finally thepolymerase process occurs when the primers are extended along the singlestrand of DNA with a thermostable enzyme. The sample undergoes the samethermal cycling and chemical reaction as it passes through each thermalcycle of the complete device. The total number of cycles in the deviceis easily altered by an extension of block length and tubing.

In another embodiment the droplets are created and/or merged on chipfollowed by their storage either on the same chip or another chip or offchip in some type of storage vessel such as a PCR tube. The chip orstorage vessel containing the droplets is then cycled in its entirety toachieve the desired PCR heating and cooling cycles.

In another embodiment the droplets are collected in a chamber where thedensity difference between the droplets and the surrounding oil allowsfor the oil to be rapidly exchanged without removing the droplets. Thetemperature of the droplets can then be rapidly changed by exchange ofthe oil in the vessel for oil of a different temperature. This techniqueis broadly useful with two and three step temperature cycling or anyother sequence of temperatures.

The invention is not limited by the method used to thermocycle thedroplets. Any method of thermocycling the droplets may be used.

Target Detection

After amplification, droplets are flowed to a detection module fordetection of amplification products. For embodiments in which thedroplets are thermally cycled off-chip, the droplets requirere-injection into either a second fluidic circuit for read-out—that mayor may not reside on the same chip as the fluidic circuit or circuitsfor droplet generation—or in certain embodiments the droplets may bereinjected for read-out back into the original fluidic circuit used fordroplet generation. The droplets may be individually analyzed anddetected using any methods known in the art, such as detecting thepresence or amount of a reporter. Generally, the detection module is incommunication with one or more detection apparatuses. The detectionapparatuses can be optical or electrical detectors or combinationsthereof. Examples of suitable detection apparatuses include opticalwaveguides, microscopes, diodes, light stimulating devices, (e.g.,lasers), photo multiplier tubes, and processors (e.g., computers andsoftware), and combinations thereof, which cooperate to detect a signalrepresentative of a characteristic, marker, or reporter, and todetermine and direct the measurement or the sorting action at a sortingmodule. Further description of detection modules and methods ofdetecting amplification products in droplets are shown in Link et al.(U.S. patent application numbers 2008/0014589, 2008/0003142, and2010/0137163) and European publication number EP2047910 to RaindanceTechnologies Inc.

In certain embodiments, amplified target are detected using detectablylabeled probes. In particular embodiments, the detectably labeled probesare optically labeled probes, such as fluorescently labeled probes.Examples of fluorescent labels include, but are not limited to, Attodyes, 4-acetamido-4′-isothiocyanatostilbene-2,2′ disulfonic acid;acridine and derivatives: acridine, acridine isothiocyanate;5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS);4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate;N-(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; BrilliantYellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin(AMC, Coumarin 120), 7-amino-4-trifluoromethylcouluarin (Coumaran 151);cyanine dyes; cyanosine; 4′,6-diaminidino-2-phenylindole (DAPI);5′5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin;diethylenetriamine pentaacetate;4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid;4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid;5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride);4-dimethylaminophenylazophenyl-4′-isothiocyanate (DABITC); eosin andderivatives; eosin, eosin isothiocyanate, erythrosin and derivatives;erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein andderivatives; 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein, fluorescein,fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144;IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneorthocresolphthalein; nitrotyrosine; pararosaniline; Phenol Red;B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene,pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; ReactiveRed 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives:6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissaminerhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red);N,N,N′,N′ tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine;tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid;terbium chelate derivatives; Cy3; Cy5; Cy5.5; Cy7; IRD 700; IRD 800; LaJolta Blue; phthalo cyanine; and naphthalo cyanine. Preferredfluorescent labels are FAM and VIC™ (from Applied Biosystems). Labelsother than fluorescent labels are contemplated by the invention,including other optically-detectable labels.

In certain aspects, the droplets of the invention contain a plurality ofdetectable probes that hybridize to amplicons produced in the droplets.Members of the plurality of probes can each include the same detectablelabel, or a different detectable label. The plurality of probes can alsoinclude one or more groups of probes at varying concentration. Thegroups of probes at varying concentrations can include the samedetectable label which vary in intensity, due to varying probeconcentrations.

In a separate embodiment the detection can occur by the scanning ofdroplets confined to a monolayer in a storage device that is transparentto the wavelengths or method or detection. Droplets stored in thisfashion can be scanned either by the movement of the storage device bythe scanner or the movement of the scanner over the storage device.

The invention is not limited to the TaqMan assay, as described above,but rather the invention encompasses the use of all fluorogenic DNAhybridization probes, such as molecular beacons, Solaris probes,scorpion probes, and any other probes that function by sequence specificrecognition of target DNA by hybridization and result in increasedfluorescence on amplification of the target sequence.

Digital PCR Performance in Droplets

Digital PCR performance in the emulsion format was validated bymeasuring a serial dilution of a reference gene, branched chain ketoacid dehydrogenase E1 (BCKDHA). Mixtures of the PCR master mix, 1×primers and probe for BCKDHA, and varying concentrations of a mixture ofhuman genomic DNA (1:1 NA14091 and NA13705) were compartmentalized intoover one million 5.3 μL droplets in a water-in-fluorinated oil emulsionusing the droplet generation microfluidic chip. The emulsion wasthermally cycled off-chip and afterwards the fluorescence of eachdroplet was analyzed by fluorescence in the readout chip (see FIG. 3).

An exemplary microfluidic system for droplet generation and readout isdepicted in FIG. 3. The microfluidic system for droplet generation andreadout. As shown in FIG. 3 a (droplet generation chip), a continuousaqueous phase containing the PCR master mix, primers, and probes, andtemplate DNA flowed into the fluidic intersection from the left, and thecarrier oil entered from the top and bottom. An emerging bolus ofaqueous liquid was imaged inside the intersection just prior to snappingoff into a discrete 4 pL droplet as the fluidic strain began to exceedthe surface tension of the aqueous liquid. The steady train of dropletsleaving the intersection toward the right was collected off chip as astable emulsion for thermal cycling. FIG. 3 b depicts the dropletspacing for readout. Flows were arranged as in 3 a, except instead of acontinuous phase, the emulsion from (a) was injected from the left intothe intersection after thermal cycling. The oil drained from theemulsion during off-chip handling, hence the emulsion appeared tightlypacked in the image before the intersection. The oil introduced in theintersection separated the droplets and the fluorescence of each dropletwas measured at the location marked by the arrow. FIG. 3 c depicts acartoon of droplet readout by fluorescence. The relatively infrequentPCR(+) droplets (light gray) flow along with the majority of PCR(−)droplets (dark gray) toward the detector. The droplets were interrogatedsequentially by laser induced fluorescence while passing through thedetection region.

In a serial dilution the average number of target DNA molecules perdroplet—called the “occupancy” from this point forward—should decreasein direct proportion to the DNA concentration. The occupancy wascalculated from Poisson statistics using the following equation wellknown to those experienced in the art:

occupancy=ln((P+N)/N)  (1)

where P and N are the numbers of PCR(+) and PCR(−) dropletsrespectively.

Droplets were analyzed by fluorescence while flowing through the readoutchip to count the numbers of PCR(+) and PCR(−) droplets (see FIG. 3 c).As each droplet passed the detection zone (marked with an arrow in FIG.3 b), a burst of fluorescence was observed. To account for smallrun-to-run differences in the fluorescence intensity that can occur dueto different chip positioning, etc., each set of data was scaled suchthat the average fluorescence intensity of the empty droplets was 0.1 V.FIG. 4 a shows a very short duration of a typical trace of fluorescencebursts from individual droplets for the sample with the highest DNAconcentration in the series. PCR(+) and PCR(−) droplets were easilydiscriminated by fluorescence intensity. The two large bursts offluorescence peaking at −0.8 V arose from the PCR(+) droplets, whereasthe smaller bursts due to incomplete fluorescence quenching in thePCR(−) droplets peaked at −0.1 V. A histogram of peak intensities fromthe complete data set revealed two clear populations centered at 0.10and 0.78 V (FIG. 4 b), demonstrating that the trend evident in the shorttrace in FIG. 4 a was stable over much longer periods of time.Integration over the two populations in FIG. 4 b yielded a total of197,507 PCR(+) and 1,240,126 PCR(−) droplets. Hence the occupancy was0.15 for this sample by Eqn. 1, corresponding to the expected occupancyof 0.18 based on the measured DNA concentration of 110 ng/μL. Theoccupancy was measured for each sample in the serial dilution and fit tothe dilution equation:

occupancy(n)=A/f ^(n)  (2)

where n is the number of dilutions, A is the occupancy at the startingconcentration (n=0), and f is the dilution factor. The linear fit was inexcellent agreement with the data, with an R² value of 0.9999 and thefitted dilution factor of 4.8 in close agreement with the expected valueof 5.0.

Multiplexing Primers in a Digital PCR Reaction

Droplet based digital PCR technology, as described in Link et al. (U.S.patent application numbers 2008/0014589, 2008/0003142, and2010/0137163), Anderson et al. (U.S. Pat. No. 7,041,481 and whichreissued as U.S. Pat. No. RE 41,780) and European publication numberEP2047910 to Raindance Technologies Inc, (the contents of each of whichare incorporated by reference herein in their entireties) utilizes asingle primer pair per library droplet. This library droplet is mergedwith a template droplet which contains all the PCR reagents includinggenomic DNA except for the primers. After merging of the template andthe primer library droplets the new droplet now contains all thereagents necessary to perform PCR. The droplet is then thermal cycled toproduce amplicons. In one embodiment, the template DNA is diluted in thetemplate mix such that on average there is less than one haploid genomeper droplet.

Having only one haploid genome (i.e., one allele) per droplet givesdroplet PCR advantages over standard singleplex or multiplex PCR intubes or microwells. For example, in traditional PCR, both alleles arepresent in the reaction mix so if there is a difference in the PCRefficiency between alleles, the allele with the highest efficiency willbe over represented. Additionally, there can be variances in thesequence to which the PCR primers hybridize, despite careful primerdesign. A variance in the primer hybridization sequence can cause thatprimer to have a lower efficiency for hybridization for the allele thathas the variance compared to the allele that has the wild type sequence.This can also cause one allele to be amplified preferentially over theother allele if both alleles are present in the same reaction mix.

These issues are avoided in droplet based PCR because there is only onetemplate molecule per droplet, and thus one allele per droplet. Thus,even if primer variance exists that reduces the PCR efficiency for oneallele, there is no competition between alleles because the alleles areseparated and thus uniformly amplified.

Optimization of traditional multiplexing of standard PCR primers intubes or wells is known to be difficult. Multiple PCR amplicons beinggenerated in the same reaction can lead to competition between ampliconsthat have differing efficiencies due to differences in sequence orlength. This results in varying yields between competing amplicons whichcan result in non uniform amplicon yields. However, because dropletbased digital PCR utilizes only one template molecule per droplet, evenif there are multiple PCR primer pairs present in the droplet, only oneprimer pair will be active. Since only one amplicon is being generatedper droplet, there is no competition between amplicons, resulting in amore uniform amplicon yield between different amplicons.

A certain amount of DNA is required to generate either a specificquantity of DNA and/or a specific number of PCR positive droplets toachieve sufficient sequencing coverage per base. Because only apercentage of the droplets are PCR positive, approximately 1 in 3 in thestandard procedure, it takes more DNA to achieve the equivalent PCRyield per template DNA molecule. The number of PCR positive droplets andthus the amplicon yield can be increased by adding more genomic DNA. Forinstance, increasing the amount of genomic DNA twofold while maintainingthe number of droplets constant will double the amplicon yield. Howeverthere is a limit to the amount of genomic DNA that can be added beforethere is a significant chance of having both alleles for a gene in thesame droplet, thereby eliminating the advantage of droplet PCR forovercoming allele specific PCR and resulting in allelic dropout.

One way to allow the input of more genomic DNA is by generating moredroplets to keep the haploid molecules per droplet ratio constant. Forinstance doubling the amount of DNA and doubling the amount of dropletsincreases the amplicon yield by 2× while maintaining the same haploidgenome per droplet ratio. However, while doubling the number of dropletsisn't problematic, increasing the amount of DNA can be challenging tousers that have a limited amount of DNA.

The multiplexing of PCR primers in droplets enables the simultaneousincrease in the number of PCR droplets while keeping the amount of inputDNA the same or lower to generate an equal or greater amplicon yield.This results in an overall increase in the amount of PCR positivedroplets and amplicon yield without the consumption of more DNA.

By way of example, if there is an average of 1 haploid genome per every4 droplets or ¼ of the haploid genome per droplet and one PCR primerpair per droplet, the chances of the correct template being present forthe PCR primer in the droplet is 1 out of 4. However, if there are 2 PCRprimer pairs per droplet, then there is double the chance that therewill be the correct template present in the droplet. This results in 1out of 2 droplets being PCR positive which doubles the amplicon yieldwithout doubling the input DNA. If the number of droplets containing the2× multiplexed primers is doubled and the DNA kept constant, then thenumber of PCR positive droplets drops back to 1 in 4, but the totalnumber of PCR droplets remains the same because the number of dropletshave been doubled. If the multiplexing level in each droplet isincreased to 4× and the input DNA is the same, the chance of the correcttemplate molecule being present in each droplet doubles. This results inthe number of PCR positive droplets being increased to 1 in 2 whichdoubles the amount of amplicon yield without increasing the amount ofinput DNA. Thus, by increasing the multiplexing of PCR primers in eachdroplet and by increasing the number of droplets overall, the ampliconyield can be increased by 4-fold without increasing the amount of inputDNA.

Alternatively, if the amplicon yield is already sufficient, byincreasing the multiplexing level for the PCR primers in each droplet,the amount of input genomic DNA can be dropped without sacrificingamplicon yield. For example if the multiplexing level of the PCR primersgoes from 1× to 2×, the amount of input genomic DNA can be decreased by2× while still maintaining the same overall amplicon yield.

Even though the number of PCR primer pairs per droplet is greater thanone, there is still only one template molecule per droplet and thusthere is only one primer pair per droplet that is being utilized at onetime. This means that the advantages of droplet PCR for eliminating biasfrom either allele specific PCR or competition between differentamplicons is maintained.

An example demonstration of droplet-based amplification and detection ofmultiple target sequences in a single droplet is shown here. Multiplecopies of 5 sets of primers (primers for TERT, RNaseP, E1a, SMN1 andSMN2) were encapsulated in a single droplet at various concentrationsalong with the template DNA and the PCR master mix. Probes thatspecifically bind to TERT, RNaseP, E1a, SMN1 or SMN2 were alsoencapsulated in the droplets containing the primers. Probes for TERT,RNaseP and E1a were labeled with the VIC dye and probes for SMN1 andSMN2 were labeled with the FAM dye. The sequences for TERT RNaseP, E1a,SMN1 and SMN2 were amplified by PCR. The PCR was conducted with astandard thermal cycling setting. For example:

95° C. for 10 min 31 cycles 92° C. for 15 s 60° C. for 60 s

At the end of the PCR, the fluorescence emission from each droplet wasdetermined and plotted on a scattered plot based on its wavelength andintensity. Six clusters, each representing droplets having thecorresponding fluorescence wavelength and intensity were shown. TheTERT, RNaseP and E1a clusters showed the fluorescence of the VIC dye atthree distinct intensities and SMN1 and SMN1 clusters showed thefluorescence of the FAM dye at two distinct intensities (FIG. 5). Thenumber of droplets, each having one or more sequences selected fromTERT, RNaseP, E1a, SMN1 and SMN2, can be determined from the scatteredplot.

In an another demonstration of droplet-based amplification and detectionof multiple target sequences in a single droplet, 5 sets of primers(primers for TERT, RNaseP, E1a, 815A and 815G) were encapsulated in asingle droplet at various concentrations along with the template DNA,the PCR master mix, and the probes. The five different probes TERT,RNaseP, E1a, 815A and 815G were also encapsulated in the dropletscontaining the primers. Probes for TERT and 815A were labeled with theVIC dye and probes for 815G were labeled with the FAM dye. For each ofRNaseP and E1a, two probes, one labeled with the VIC dye and the otherlabeled with the FAM dye, were encapsulated.

The droplets containing both the primers and probes were fused withdroplets containing the template. PCR reactions were conducted with thefused droplets to amply the sequences for TERT, RNaseP, E1a, 815A and815G. The PCR was conducted with a standard thermal cycling setting.

At the end of the PCR, the fluorescence emission from each fused dropletwas determined and plotted on a scattered plot based on its wavelengthand intensity. Six clusters, each representing droplets having thecorresponding fluorescence wavelength and intensity were shown. The TERTand 815A clusters showed the fluorescence of the VIC dye at two distinctintensities; the 815G clusters showed the fluorescence of the FAM dye;and the RNaseP and E1a clusters showed the fluorescence of both the FAMand the VIC dye at distinct intensities (FIG. 6). The number ofdroplets, each having one or more sequences selected from TERT, RNaseP,E1a, 815A and 815G, can be determined from the scattered plot. The copynumber of RNaseP, E1a, 815A and 815G in the template were determined bythe ratio between the number of droplets having the RNaseP, E1a, 815Aand/or 815G sequences and the number of droplets having the TERTsequence (FIG. 6).

In yet another exemplary demonstration of multiplexed primer pairs in adroplet-based digital PCR reaction, two droplet libraries weregenerated: droplet library A was generated where each droplet containedonly one primer pair; and droplet library B was generated where theprimer pairs were multiplexed at 5× level in each droplet. HapMap sampleNA18858 was processed in duplicate with droplet libraries A or B usingstandard procedures. Two μg sample DNA was used for droplet library Aand one μg sample DNA was used for the 5× multiplex droplet library B.After PCR amplification, both droplet libraries were broken and purifiedover a Qiagen MinElute column and then run on an Agilent Bioanalyzer.Samples were sequenced by Illumina on the Illumina GAII with 50nucleotide reads and the sequencing results were analyzed using thestandard sequencing metrics. The results from the 5× multiplexed dropletlibrary B were compared to the singleplex droplet library A usingstandard metrics shown in the Table below.

The results obtained from the 5× multiplexed droplet library B wereequivalent or better than what was obtained from droplet library A. Themultiplexing of primers delivers the same sequencing results for basecoverage, specificity and uniformity that the singleplexing does withthe added advantage of reduced input DNA.

Mean Base coverage Total Mapped base (0.2× Sample reads readsSpecificity coverage C1 C20 C100 of mean) Library 27431697 99.4% 0.8131394 99.5% 99.0% 98.2% 92.8% A with sample 1 Library 15147288 99.4%0.862  819 99.1% 98.2% 87.6% 78.0% A with sample 2 Library 2786137899.5% 0.847 1472 99.7% 99.3% 97.6% 89.9% B with sample 1 Library25758406 99.1% 0.837 1321 99.8% 99.4% 97.9% 91.3% B with sample 2 Totalreads: total number of sequencing read found within the provided sampledata. Mapped reads (%): percentage of total reads that mapped to thehuman genome. Specificity: percentage of mapped reads that include thetarget. The target includes all amplicon sequences with primer sequencesexcluded. Mean base coverage: average base coverage within the target.The target includes all amplicon sequences with primer sequencesexcluded. C1: % of target that has at least l× base coverage. Note:non-unique sequencing reads are mapped randomly. C20: % of target thathas at least 20× base coverage. C100: % of target that has at least 100×base coverage. Base coverage (0.2× of mean): % of target that has atleast 20% of mean base coverage.

Monochromatic Gene Copy Number Assay

Traditional digital PCR methods involve the use of a single labeledprobe specific for an individual target. FIG. 7 is a schematic depictingone-color detection of a target sequence using droplet based digitalPCR. As shown in Panel A of FIG. 7, a template DNA is amplified with aforward primer (F1) and a reverse primer (R1). Probe (P1) labeled with afluorophore of color 1 binds to the target genetic sequence (target 1).Microdroplets are made of diluted solution of template DNA underconditions of limiting or terminal dilution. Droplets containing thetarget sequence emit fluorescence and are detected by laser (Panels Band C). The number of microcapsules either containing or not containingthe target sequence is shown in a histogram (D) and quantified (E).

FIG. 8 is a schematic depicting two-color detection of two geneticsequences with a microfluidic device. As shown in Panel A of FIG. 8, atemplate DNA is amplified with two sets of primers: forward primer (F1)and a reverse primer (R1), and forward primer (F2) and a reverse primer(R2). Probe (P1) labeled with a fluorophore of color 1 binds to thetarget 1 and probe (P2) labeled with a fluorophore of color 2 binds tothe target 2 (Panels B and C). Droplets are made of diluted solution oftemplate DNA under conditions of limiting or terminal dilution. Dropletscontaining the target sequence 1 or 2 emit fluorescence of color 1 or 2respectively and are optically detected by laser (Panels B and C). Thenumber of microcapsules containing target 1 or 2 is shown by histogramin Panel D.

Methods of the invention involve performing accurate quantitation ofmultiple different DNA targets by dPCR using probes with the samefluorophore. FIG. 9 is a schematic depicting two-color detection ofthree genetic sequences with a microfluidic device. As shown in Panel Aof FIG. 9, a template DNA is amplified with three sets of primers:forward primers (F1, F2 and F3) and reverse primers (R1, R2 and R3).Probes (P1, P2 and P3) are labeled with fluorophores (color 1, color 2and color 1) and bind to the target genetic sequences (target 1, target2 and target 3) (Panels B and C). Microdroplets are made of dilutedsolution of template DNA under conditions of limiting or terminaldilution. Microdroplets containing target sequence 1 or 3 emitfluorescence of color 1 at two different intensities; and microdropletscontaining target sequence 2 emit fluorescence of color 2. The number ofmicrodroplets containing target 1, 2 or 3 is shown by histogram in PanelD.

Recent results from the droplet digital PCR (dPCR) shows that multipleindependent PCR reactions can be run and separately quantified using thesame fluorophore. Specifically, an SMN2 assay yields an unexpectedpopulation of droplets with slightly elevated signal in the FAMdetection channel.

The results are depicted in FIG. 10. The left-side dot plot in FIG. 10depicts the effect of having the SMN1 blocker present in the reaction.The four clusters depicted in the left-side dot plot are as follows: thetop left cluster includes microdroplets containing the referencesequence (SMARCC1); the bottom left cluster includes microdroplets notcontaining any sequence; the bottom middle cluster includesmicrodroplets containing sequence for SMN1; and the bottom right clusterincludes microdroplets containing sequence for SMN2. The dot plot on theright-side of FIG. 10 depicts four clusters where no SMN1 blocker waspresent in the reaction: the top left cluster includes microdropletscontaining the reference sequence (SMARCC1); the bottom left clusterincludes microdroplets not containing any sequence; the bottom middlecluster includes microdroplets containing sequence for SMN1; and thebottom right cluster includes microdroplets containing sequence forSMN2. The shift of the bottom middle cluster in right panel as comparedto left panel confirms that fluorescence intensity provides a verysensitive measurement for the presence of a sequence.

Without intending to be bound by any theory, the simplest explanation isthat the cluster arises from weak association of the SMN2 probe to theSMN1 gene despite the presence of a blocker to that gene (anonfluorescent complementary probe to the SMN1 gene).

One definitive confirmation of SMN1 as the source of the unexpectedcluster was an observed dependence of the intensity of this feature onthe presence of the SMN1 blocker. A clear shift toward higher FAMfluorescent intensities was observed in the absence of the blocker (FIG.10). In another definitive confirmation the ratio of the SMN1 (putative)population size to the reference size of 0.96 in perfect agreement withexpectation (two copies of each) (S-131 sample). Another sample, S-122,with the same number of SMN1 copies yielded a ratio of 0.88 in one runand 0.93 in another, also consistent with the proposed explanation ofthe unexpected cluster.

Without intending to be bound by any theory, these observations indicatethat SMN2 probe binding to SMN1 DNA yields an elevated fluorescentsignal. A simple kinetic model explaining this phenomenon assumes thatthe hybridization of the SMN2 probe to the SMN1 DNA achieves equilibriumat a faster rate than the polymerase fills in the complementary strand.The amount of probe fluorophore that is released in each thermal cycleis therefore proportional to (or even equal to) the number of boundprobes. Thus the lower the binding affinity the fewer the number ofprobe fluorophores that are released. Due to SMN1 sequence mismatch(es)with the SMN2 probe, the affinity of the probe is certainly expected tobe lower to SMN1 than SMN2. This model also explains the signaldependence on the sMN1 blocker: the blocker competitively inhibits theSMN2 probe hydrolysis by the polymerase exonuclease activity.

It may also be, however, that the probe hybridization does not reachequilibrium before exonuclease activity. In this case, the associationrates would play a more dominant role. Similar logic applies. Thebinding rate to the matching site is likely to be faster than to themismatch site, and the blocker would act to decelerate probe binding tothe mismatch site. The binding of SMN2 probe to SMN1 DNA might bedetectable by conventional bulk qPCR, especially in absence of SMN2, buthighly quantitative results like those shown here are very unlikely.Definitely, there is no report of qPCR or any other techniquequantifying two different DNA sequence motifs with the same colorfluorophore. Sequestration of the individual reactions by singlemolecule amplification within droplets eliminates any confusionregarding mixed contributions to the signal.

The advantage of quantifying DNA with multiple probes of the same colorfluorophore extends beyond the example of two highly homologoussequences shown here. Rather, any plurality of sequences of any degreeof similarity or dissimilarity can be quantified so long as thedifferent probes have significantly different binding occupancies totheir respective DNA binding sites.

Another advantage of the dPCR approach for multiplexed reactions is thatthe different reactions do not compete with each other for reagents asthey would in a bulk qPCR assay. However, the possibility for unintendedcross-reactivity remains. A multiplexes assay can require a more dilutesample. For instance, at 10% occupancy a duplex reaction would havedouble occupancy 1% of the time. Hence 1 in 10 PCR+ droplets would bedoubles, resulting in a final intensity at least as high and possiblyhigher than the brighter of the two probes. For a simple duplex systemthe contribution from each probe could be recovered. In this example thetotal number of PCR+ droplets for probe 1 would be (Probe1)+(Probe1+Probe2). Higher degrees of multiplexing would require greaterdilution. For example, for a 4-plex at 1% occupancy the probability ofone probe overlapping any of the other 3 is ^(˜)3%, and that error maybe too high for some applications. The need for large dilutions stronglyfavors the large number of dPCR reactions.

In another example of the invention, a single fluorophore (FAM) was usedin a gene copy number assay for both the reference and the target DNA. Amodel system was used with varying concentrations of plasmid DNA torepresent a change in the target gene copy number, relative to areference gene, equivalent to 0-16 copies of the target gene per cell.BCKDHA and SMN2 plasmid DNA served as the reference and target with 1×and 0.5× primers and probes respectively. With a starting ratio of 8:1SMN2 to BCKDHA, the sample was diluted serially by 2× into a solution ofBCKDHA at the same concentration to vary just the amount of SMN2. Theresultant samples were emulsified, thermally cycled, and over 105droplets were analyzed for each sample as described in the previoussection. The process was repeated in triplicate.

Methods of the invention also include analytical techniques foridentification of fluorescence signatures unique to each probe. In thisexample of the invention, histograms of the droplet fluorescenceintensities are shown in FIG. 11 a for three different template DNAsamples: a no template control (dotted line), BCKDHA only (solid line),and 1:1 BCKDHA to SMN2 (dashed line). For clarity, the histograms areshown both overlapped to highlight the similarity for certain peaks, andoffset from each other to reveal all of the features. In the case of 1:1BCKDHA to SMN2, three populations were readily apparent: a dominantfeature appeared at 0.08 V, and two smaller peaks were evident at 0.27and 0.71 V. The dominant feature at 0.08 V was assigned to PCR(−)droplets since both small peaks disappeared, but the large one remained,in the no template control. The peak at 0.71 V was assigned to BCKDHAsince it was the sole feature arising with the addition of just BCKDHA,and the peak at 0.27 V appeared on subsequent addition of SMN2,completing the assignments. A very small peak appeared at ˜0.9 V, notvisible on the scale of FIG. 11 a, that corresponded to dropletsoccupied by both genes. As another method of the invention, once thedifferent peaks are identified, droplets within each peak were countedcorresponding to each possible state (PCR(+) for either BCKDHA or SMN2,or both, or PCR(−)), and the gene copy number was then determined fromthe ratio of occupancies. Gene copy numbers for each sample in theserial dilution are plotted in FIG. 11 b against expected values(observed ratios of SMN2 to BCKDHA to expected ratios of SMN2 toBSKDHA), with an excellent linear fit (y=1.01×) across the full range(R2=0.9997, slope=1.01), demonstrating accurate and precise measurementof the equivalent of 0 to 16 copies of SMN2 per cell.

Detection of Alternatively Spliced Transcripts

The same principle can be used to detect and count alternatively splicedtranscripts. TaqMan assays can be designed that are specific for each ofthe exons in an RNA transcript. After the RNA is turned into cDNA it canbe encapsulated into a droplet at 1 copy or less per droplet. Thedroplet would also contain the multiplexed TaqMan assay for each of theexons. Each of the TaqMan assays would contain a different probe but allthe probes would have the same fluorescent dye attached. The dropletswould be thermocycled to generate signal for each of the TaqMan assays.If there are multiple splice variants in the sample they each willcontain a different number of exons depending on the splicing events.The fluorescent intensity of each droplet would be different dependingon the number of exons present. By counting the number of droplets withdifferent intensities it would be possible to identify the presence andabundance of different splice variants in a sample.

Copy Number Variants in a Heterogeneous Sample

It would be possible to determine if a heterogeneous sample containedcomponents with different copy level numbers. If the copy numbervariants to be assayed were spaced close enough along the chromosome,the DNA from a sample could be fragmented and encapsulated in dropletsat a level of one haploid genomic equivalent or less per droplet. Thedroplet would also contain a TaqMan assay specific for the copy numbervariant. The intensity of the signal in each droplet would depend on thenumber of copy number variants are present for the sample. Counting ofthe number of droplets of different intensities would indicate thingslike how many cells in a particular sample had what level of copy numbervariants.

Tuning Taqman® Probe Fluorescence Intensity

Identifying probes by fluorescence intensity often requires adjustingthe brightness of the probes, particularly for higher-plex assays withdense probe patterns. In the previous section the probes for the genecopy number assay yielded very well resolved peaks (FIG. 11 a). Clearlyroom exists to accommodate one or multiple extra probes in the copynumber assay within the resolution of the measurement, but a method foradjusting the fluorescence intensity of the new probes is required toavoid interference with the existing assay. One method of the inventioninvolves varying the probe and primer concentrations together as a verysimple technique to optimize relative intensities in higher-plexreactions.

FIG. 12 is a schematic for tuning the intensity of a detectable label toa particular target with a microfluidic device. As shown in Panel A ofFIG. 12, a template DNA is amplified with two sets of primers: forwardprimers (F1 and F2) and reverse primers (R1 and R2). Probes (P1 and P2)are labeled with fluorophore of color 1 and bind to target 1 and target2 respectively. Fluorescence from target 2 is lower in intensity thanthat from target 1 due to single base mismatch between P2 and target 2.As shown in Panel B, template DNA is amplified with two sets of primers:forward primers (F1 and F2) and reverse primers (R1 and R2) (Panel B).Fluorescence from target 2 is lower in intensity than that from target 1due to the presence of a competing probe 2 that is not labeled with thefluorophore. As shown in Panel C, template DNA is amplified with twosets of primers: forward primers (F1 and F2) and reverse primers (R1 andR2). Probes (P1 and P2) are labeled with fluorophore of color 1 and bindto target 1 and target 2 respectively. Fluorescence from target 2 islower in intensity than that from target 1 due to the presence of acompeting probe 2 that is labeled with a different fluorophore.

FIG. 13 shows probe fluorescence intensities throughout a serialdilution of the probes and primers for a different reference gene,ribonuclease P (RNaseP), against a constant amount of genomic DNA fromthe Coriell cell line NA3814 at an occupancy of 0.02 target DNAmolecules per droplet. The probe fluorescent intensities varied indirect proportion to probe concentration over a narrow concentrationrange spanning ˜0.15 to 0.4 μM (R²=0.995)—roughly centered about thetypical probe concentration of 0.2 μM—after compensation for dilutionerrors and other run-to-run differences such as optical realignmentsusing the intensity of the PCR(−) droplets as a reference. In summary,probe intensities can be varied by dilution over a small but adequaterange for the purpose of tuning multiplexed assays without affecting theamplification itself.

Although the example above for adjusting probe fluorescence intensitiesinvolves varying probe and primer concentrations together by the samefactor, the invention is not limited to this method alone for varyingprobe intensity. Other methods known to those familiar with the art forvarying probe intensities are also considered. Such methods includevarying just the probe concentration; varying just the primerconcentrations; varying just the forward primer concentration; varyingjust the reverse primer concentration; varying the probe, forward, andreverse primers concentrations in any way; varying the thermal cyclingprogram; varying the PCR master mix; incorporating into the assay somefraction of probes that lack fluorophores; or incorporating into theassay any hybridization-based competitive inhibitors to probe binding,such as blocking oligomer nucleotides, peptide nucleic acids, and lockednucleic acids. The invention incorporates the use of these methodsadjusting probe fluorescence intensity, or any other methods foradjusting probe fluorescence intensity, used either by themselves or inany combination.

Higher-Plex Reactions

One method of the invention involves performing higher-plex assays witha single probe color (i.e. fluorophore). As described above, probefluorescent intensities can be adjusted by a variety of means such thateach intensity level uniquely identifies a DNA target. For example,targets T1, T2, T3, and T4 might be uniquely identified by intensitylevels I1, I2, I3, and I4. Not intending to be bound by theory, themaximum number of intensity levels possible for unique identification oftargets is related to the resolution of the different intensitylevels—that is the spread of intensities for each particular probecompared to the separation between the average intensities of theprobes—and it is also related to the intensity of the empty dropletsthat tends to grow with increasing numbers of probes. The number ofintensity levels can be 0, or 1, or 2, or 3, or 4, or up to 10, or up to20, or up to 50, or up to 100. The number of intensity levels can behigher than 100. In the examples show below, as many as three intensitylevels are demonstrated.

Another method of the invention involves performing higher-plex assaysusing multiple different probe colors (i.e. fluorophores). As above forthe monochromatic multiplexing assay, for each color probe, multipletargets can be identified based on intensity. Additionally, multiplecolors that are spectrally separable can be used simultaneously. Forexample, a single droplet might contain four different probes formeasuring four different targets. Two probes might be of color A withdifferent intensities (say, A1 and A2), and the other two probes ofcolor B with different intensities (say B1 and B2). The correspondingtargets are T1, T2, T3, and T4 for A1, A2, B1, and B2 respectively. If adroplet shows an increase in fluoresce in color A, the droplet thereforecontained either targets T1 or T2. Then, based on the fluorescenceintensity of color A, the target could be identified as T1 or the targetcould be identified as T2. If, however, a droplet shows an increase influorescence in color B, the droplet therefore contained either targetsT3 or T4. Then, based on the fluorescence intensity of color B, thetarget could be identified as T3 or the target could be identified asT4. Not intending to be bound by theory, the maximum number of differentcolors possible is limited by spectral overlap between fluorescenceemission of the different fluorophores. The maximum number of colors canbe 1, or 2, or 3, or 4, or up to 10, or up to 20. The maximum number ofcolors can be higher than 20. In the demonstrations that follow, thelargest number of colors is two.

Another method of the invention involves performing higher-plex assaysusing multiple different probe colors (i.e. fluorophores), howeverunlike the strategy above where each target is identified by single typeof probe with a unique color and intensity, instead in this method asingle target may be identified by multiple probes that constitute aunique signature of both colors and intensities. For example, a singledroplet might contain four different probes for measuring threedifferent targets (say, T1, T2, and T3). Two probes might be of color A(say, A1, and A2), and two probes might be of color B (say, B1 and B2).T1 is measured by probe A1, T2 is measured by probe B1, but T3 ismeasured by both probes A2 and B2. Thus, when a droplet contains T1 onlyincreased fluorescence appears in color A. When a droplet contains T2only increased fluorescence appears in color B. However when a dropletcontains T3, increased fluorescence appears in both colors A and B.

Generally, without wishing to be constrained by theory, the above threemethods for higher-plex dPCR are simplest to implement under conditionsof terminal dilution, that is when the probability of multiple differenttarget molecules co-occupying the same droplet is very low compared tothe probability of any single target occupying a droplet. With multipleoccupancy arises the complexity of simultaneous assays competing withinthe same reaction droplet, and also complexity of assigning theresulting fluorescence intensity that involves a combination offluorescence from two different reaction products that may or may not beequal to the sum of the two fluorescence intensities of the individualreaction products. However, methods of the invention can accommodatethese complications arising from multiple occupancy.

Methods of the invention for higher-plex reactions also include methodsfor primer and probe pairing. In the simplest case targets are unlikelyto reside on the same DNA fragments, such as when targets are fromdifferent cells; or when targets are from different chromosomes within asingle cell type; or when targets are distant from each other within asingle chromosome such that they become physically separated during DNAfragmentation; or when targets are very close to each other within achromosome, but nevertheless become separated by targeted cleavage ofthe DNA, such as by restriction enzyme digestion; or for any otherreason. In such cases each probe can be paired with a single set ofprimers (forward and reverse). However, in other cases the targetregions might frequently reside on the same DNA fragments, for examplewhen targets reside within the same codon, or for any other reason. Insuch cases, a single set of primers might serve for multiple probes (foran example, see Pekin et al.).

Higher multiplex reactions can be performed to distinguish thehaplotypes of two SNPs. For example, assume that at position one therecan be genotypes A or A′ and at position two there can be genotypes of Bor B′. In a diploid genome four unique haplotypes are possible (A,B;A,B′; A′,B; and A′,B′). If for example A′ and B′ represent drugresistant mutations for infection, it is often the case that A′B and AB′are less sever and treated differently than A′B′ which represents asignificant drug resistance that must be treated with extreme care.Digital PCR with intensity discrimination is ideally suited foridentifying low prevalence of A′B′ in a background of mixtures of theother three haplotypes. Haplotyping information is also important forconstruction of haplotypes in HLA. One way that the present example canbe constructed is by assay design such that color one is used for A andis of high or low intensity indicative of allele A or A′ respectivelyand color two is used for B and is of high or low intensity respectivelyindicative of B or B′. Populations of [color1, color2] corresponding to[Low, Low] would be a measure of an allele of AB and [high, low] alleleA′B and an allele of [A′B′] will be readily distinguishable as [high,high] even in a background that is predominately a mixture of A′B andAB′. See FIG. 22. In some cases it will be advantageous to start byencapsulating into the droplets long single molecules of nucleic acidthat contain both A and B SNP location and in other cases it will bedesirable to start by encapsulating single cells, bacteria or otherorganism within the droplets prior to releasing the nucleic acid fromthe organism. In still other embodiments the multiplex intensitydetection of multiple simultaneous targets can be used as surrogatemarkers for multiple types of binding interactions or labeling of targetmaterials. This technique is also not limited to single moleculedetection and can be used for haplotype detection in single cells (e.g.,bacteria, somatic cells, etc.). In single cell analysis, a sorting stepmay be applied prior to haplotyping.

5-Plex Assay for Spinal Muscular Atrophy

An aspect of the invention was reduced to practice in an exampledemonstration of the quantitation of several genetic markers for spinalmuscular atrophy (SMA). SMA was selected for one of the exampledemonstrations due to both its important clinical significance as wellas its complicated genetics. It is the second-most prevalent fatalneurodegenerative disease and affects ^(˜)1 in 10,000 live births. SMAis most often caused by homozygous absence of exon 7 within the survivalof motor neuron 1 gene (SMN1, reviewed by Wirth et al.), however theseverity of the condition is modulated by the number of gene copies ofSMN2 with prognosis ranging from lethal to asymptomatic over 1-5 copynumbers (reviewed by Elsheikh et al.). Hence accurate quantitation ofSMN2 copy number is important for clinical prognosis and geneticcounseling. Aside from large deletions of SMN1, a number of single pointmutations or short deletions/duplications within the same gene alsoaccount for ^(˜)4% of cases of SMA. In a significant step toward acomprehensive SMA assay, the multiplexed dPCR assay demonstrated herecontains both copy number assays (for SMN1 & 2) and an assay for one ofthe prevalent SNPs (c.815A>G).

One embodiment of the invention is a 5-plex assay for SMA diagnostics.The 5-plex assay quantifies common genetic variants impacting SMAincluding two copy number assays for the SMN1 and SMN2 genes with BCKDHAas a reference, and a SNP assay for the c.815A>G mutation. Twodifferently colored fluorophores, FAM and VIC, were used to uniquelyidentify each of the assays. The probes for SMN1 and SMN2 contained onlyFAM, and for c.815A only VIC. However, mixtures of VIC and FAM-labeledprobes were used for BCKDHA and c.815G. The use of VIC and FAMfluorophores in this example does not limit the invention, rather the5-plex assay can be used with any spectrally separable fluorophorescompatible with the TaqMan assay, or any other fluorogenichybridization-based probe chemistries. For validating the assay, a modelchromosome was synthesized containing a single target region for each ofthe different primer/probe pairs. EcoRV restriction sites flanked eachtarget, allowing separation of the fragments.

As another method of the invention, histogram-based data presentationand analysis is incorporated into the invention for identifying andcharacterizing statistically similar populations of droplets that arisefrom one probe signature (color and intensity), and for discriminatingone population of droplets from the others. FIG. 14 a shows a2-dimensional histogram of droplet fluorescence intensities as acontoured heat map, with hotter colors representing higher occurrences.Standard techniques were used to compensate for spectral overlap of theFAM and VIC signals. Samples were run at 0.006 occupancy per target. Sixpopulations were clearly evident, five for the assay and one for PCR(−)droplets. As one method of the invention, the populations were assignedby selective exclusion of assay components. For example, excluding theSMN2 primers and probe eliminated the population at the bottom right inthe histogram, but otherwise the distribution remained unchanged.Assignments are labeled in FIG. 14 a. As we have found to be generallytrue for this method of multiplexing, the assay worked immediately withwell resolved or at least distinguishable populations for each target.As another method of the invention, the relative positions of thedifferent populations in the histogram were then adjusted into aregularly spaced rectangular array by tuning the probe concentration asdescribed in the previous section. Usually no more than two iterationsare required for optimization.

In another method of the invention, the different populations weresufficiently well resolved to allow droplets within each population tobe counted by integration across rectangular boundaries. The boundarieswere positioned at mid-sections between neighboring peaks. The methodsof the invention are not constrained to rectangular boundaries, or tospecific boundary locations between peaks. Rather, any closed orunclosed boundary condition can suffice. Boundary conditions do not needto be “binary” either, in the sense that weighted integrations can alsobe performed across the boundaries to arrive at droplet counts. The peakposition of each cluster varied by no more than 2% from run to run afternormalization to the intensity of the empty droplets to account forvariations in detection efficiency (data not shown). Hence, onceidentified, the same boundaries for integration could be reused betweensamples. The methods of the invention are not limited to fixed boundarypositions. Dynamic population identification and boundary selection inbetween samples or studies is anticipated. Twenty different patientsamples from the Coriell cell repositories were analyzed with thisassay: 4 afflicted with SMA, 1 SMA carrier, and 15 negative controls.Assay results are shown in FIG. 14 b. Gene copy number was calculated asbefore, as the ratio of occupancies derived from the number of targetdroplets vs. reference droplets. Like the copy number measurement inFIG. 11, each assay yielded ratios very close to the expected integervalues, but when all of the patient data was plotted as actual ratio vs.expected integer ratio a small systematic deviation from the ideal slopeof 1 was observed. Measured slopes were 0.92, 0.92, and 0.99 for SMN1,SMN2, and c.815A respectively. For clarity, the data in FIG. 14 b wasscaled to the ideal slope of 1.

The measured genotypes of the different patients were consistent withtheir disease conditions (unafflicted, carrier, or afflicted). Thepatients afflicted with SMA each had zero copies of SMN1 (numbers SMA1-4 in FIG. 14 b), the carrier had just one copy, and the negativecontrols all had two or three copies (numbers 1-15). Three unrelatedindividuals (numbers 6, 8, and 9) had three copies of SMN1, occurring ata rate of 20% which is similar to a previous report for healthyindividuals. Variability in SMN1 copy number is not surprising since itlies within an unstable region of chromosome 5q13. A larger variety ofSMN2 copy numbers was observed. One to two copies were most common inthe control group, although one individual had zero copies, adistribution consistent with expectations for normal individuals. TheSMA carrier and afflicted patients had elevated copy numbers of SMN2 onaverage: 5 for the carrier, two afflicted with 3 copies, and the otherswith 2 copies. The afflicted patients were all diagnosed as SMA Type I,the most severe form, based on clinical observations according to theCoriell repository. The strong genotype/phenotype correlation betweenSMN2 copy number and disease severity suggests that the two individualswith three copies of SMN2 might have an improved Type II prognosis,especially for the patient SMA 1 who had survived to three years at thetime of sampling, much beyond the typical maximum life expectancy forSMA Type I of 2 years. However there remains reluctance to predictdisease outcome based on SMN2 copies alone since other less wellcharacterized or unknown modifying genes may impact prognosis andbecause not all SMN2 copies may be complete genes. Furthermore some TypeI patients have begun surviving longer in newer clinical settings.Hence, with little clinical information regarding the patients availableto us; we can conclude that our SMN2 assay results were consistent withbroad expectations for disease severity.

The SNP assay revealed that all patients carried the normal c.815Agenotype and no instances of c.815G were observed. The mutation isrelatively rare and hence was not expected to appear in a small patientpanel. Of interest, however, was the presence of an apparent extra genefragment in two unrelated individuals that was uncovered with the SNPassay. The c.815A>G assay does not discriminate between SMN1 and SMN2due to their high sequence similarity, and hence the total copies ofc.815A and G should equal the sum of the copies of SMN1 and SMN2. Thiswas true for all patients except for healthy patients number 1 and 2,both of whom had one extra copy of c.815A. c.815 lies on exon 6, and theSNP that discriminates between the SMN1 and SMN2 genes lies on exon 7,hence the extra genes may be fragments of SMN1 lacking exon 7. Thisseems reasonable because the deletion of exon 7 is the common mutationcausing 95% of cases of SMA (reviewed by Wirth et al.) and it is carriedby 1/40 to 1/60 adults. Thus these patients might have been typicalcarriers of SMA but for the acquisition of at least one compensatinghealthy copy of SMN1 on the same chromosome.

9-Plex Assay for Spinal Muscular Atrophy

A 9-plex assay for certain SMA related targets was also demonstratedwith just two colors (probes containing FAM and VIC fluorophores). Asidefrom the optimized primer and probe concentrations, assay conditions andexperimental procedures were identical to the 5-plex assay above. FIG.15 a shows the various droplet populations in 2-D histograms beforeoptimization of probe concentrations. The identity of the differenttargets is shown on the figure itself. As one method of the invention,the identification of the different populations was made as before, byselective exclusion and/or addition of one or more assays. Most of thepopulations were already well resolved, with the exception of the probefor the c.815A genotype that was in close proximity with the clustercorresponding to empty droplets. After three iterations of optimizationof probe concentrations, all of the target populations were wellresolved from each other, and well resolved from the empty droplets(FIG. 15 b). Three methods of the invention were highlighted in thisdemonstration: (1) nine DNA targets were uniquely identified in atwo-dimensional histogram, far beyond the capabilities of conventionalqPCR; (2) target DNA molecules were distinguished on the basis of somecombination of both color and intensity arising from one or multipleprobes against the same target; and (3) the relative positions of thetarget molecules within the histogram were adjusted by varying the probeconcentrations to optimize the pattern of colors and intensities forincreased resolution amongst the various droplet populations.

As one method of the invention, different droplet populations wereidentified by selective addition or exclusion of assays in the examplesabove. However the invention is not limited to this method alone.Rather, any method for population assignments known to those in the artare considered. Methods of the invention include any method that cancause an identifiable displacement, appearance, or disappearance of oneor more populations within the histograms including changing the probeand primer concentrations together, either by the same factor or bydifferent factors; changing the probe concentration alone; changing theprimer concentrations alone; changing the thermal cycling conditions;and changing the master mix composition. Another method of the inventiontakes advantage of prior knowledge of the position of an assay within ahistogram to assist assignment.

Multiplexing Capacity

The level of multiplexing demonstrated in the preceding SMA example was9×, significantly exceeding the maximum practicable number with qPCR.Without wishing to be constrained by theory, the two main limitationsare the resolution between assays and the increasing fluorescenceintensity of empty droplets with higher loading of probes. A method ofthe invention involves optimizing the pattern of colors and intensitiesof the different probes for maximum multiplexing while still achievingadequate specificity for each individual reaction. Although rectangulararrays of droplet populations were demonstrated for the 5- and 9-plexreactions, another desirable pattern is the tight-packed hexagonalarray. However the invention is not constrained to any particular arraystrategy.

Adding extra colors would increase the capability even further, howeverwith some diminishing returns because the fluorescence of the emptydroplets would continue to rise. The capacity could be yet furtherincreased with better probes yielding larger differential signals, suchas hybrid 5′-nuclease/molecular beacon probes that reduce background bycontact quenching yet exhibit the bright signals typical of freeunquenched fluorophores. With such improvements multiplexing capacityexceeding 50× can be envisioned.

Combined Multiplexing with Optical Labeling

Using droplet-based microfluidics, multiple targets can also be measuredsimultaneously by a different method. According to the alternativemethod, primers and probes can be loaded individually into dropletsalong with an optical label to uniquely identify the assay. Typicallythe optical label is a fluorophore, or a combination of differentfluorophores, that are spectrally distinct from the probe fluorophore.Various different types of droplets, each containing different assaysthat are uniquely identified by different optical labels, can be mixedinto a “library” of droplets. Then, according to methods of theinvention above, library droplets are merged one-to-one with dropletscontaining template DNA. After thermal cycling, some droplets thatcontain template DNA will exhibit brighter fluorescence at the emissionwavelengths of the probes. The specific target DNA molecules giving riseto these PCR(+) signals are subsequently identified by the opticalprobes. In one study, the six common mutations in KRAS codon 12 werescreened in parallel in a single experiment by one-to-one fusion ofdroplets containing genomic DNA with any one of seven different types ofdroplets (a seven-member library), each containing a TaqMan® probespecific for a different KRAS mutation, of wild-type KRAS, and anoptical code.

In one method of the invention, optical labeling can be combined withthe various methods for multiplexing dPCR already incorporated into thisinvention. For example, a single optical label might code for the entire5-plex SMA assay, above, instead of just a single assay as in the KRASexample above. In this manner, other optical labels might code fordifferent screening assays for newborn infants. According to othermethods of the invention, above, a single DNA sample from an infantcould then be analyzed with all of the assays simultaneously by mergingdroplets containing the DNA one-to-one with library droplets containingthe optically encoded assays.

As an example of combining multiplexing with optical labels, a so called3×3×3 combination multiplex reaction with optical labeling wasdemonstrated (3×3 optical labeling with two fluorophores, each encodinga triplex assay, for a total of 27-plex). Two fluorophores were employedfor optical labeling, Alexa633 and CF680 (excited by a 640 nm laser),with three intensity levels each producing nine total optical labels. Asbefore with the 5- and 9-plex assays for SMA, TaqMan assays were usedwith FAM and VIC fluorophores (excited by a 488 nm laser). Thefluorescence from the FAM and VIC fluorophores were recordedsimultaneously with the fluorescence from the optical labels, requiringmodifications to the optical layout of the instrumentation described forthe SMA assay (the optical schematic for two-laser excitation and4-color detection is shown in entirety in FIG. 16). Also, co-flowmicrofluidics were used in this example (the use of co-flow basedmicrofluidics for this application is one of the methods of theinvention described above). In this case, the template DNA wasintroduced into the chip in one flow, and the PCR master mix, theprimers and probes for one triplex assay, and the unique composition offluorophores for the optical label were introduced into the chip inanother flow simultaneously. The two flow streams converged in a fluidicintersection upstream from the droplet forming module, and thus eachdroplet formed contained the contents of both flow streams. Methods toimplement co-flow microfluidics are well known to those in the art. Thedroplets were collected, and then the procedure was repeated with thenext triplex assay and optical label. The procedure was repeated a totalof nine times, once for each pair of assays and optical labels. All ofthe droplets were collected into a single PCR tube and thermally cycledoff chip. The mixture of thermally cycled droplets was reinjected intothe same read-out chip as used for the SMA assay, above, and thefluorescence intensities of the assays from all four fluorophores wasrecorded.

FIG. 17 shows the cumulative results from all droplets in the 3×3×3assay using co-flow microfluidics. The figure shows two 2-D histogramsof droplet fluorescence intensities, the histogram on the left from allof the optical labels, and the histogram on the right from the assays.Standard methods were used to compensate for spectral overlap. Thehistograms are shown as a heat maps, with hotter colors designatinglarger numbers of droplets. Nine different clusters of droplets wereclearly evident in the histogram of the optical labels, corresponding toeach of the nine different optical labels: there is a small group offour clusters at the bottom left corner of the histogram, correspondingto optical labels with the lowest fluorescent intensities; and there arefive clusters appearing as linear streaks at the higher intensities. Thedroplet clusters were less distinct in the histogram for the assay, butthis was as expected because the droplets shown contained all of thetriplex assays. The individual assays became clearly distinct once asingle type of assay was selected by using the optical labels, asfollows.

Methods of the invention involve selecting individual populations ofdroplets all containing the same optical labels, or groups of opticallabels. In some methods of the invention, boundaries of fluorescenceintensity were used to specify populations. In the example shown here, arectangular boundary was used specifying the minimum and maximumfluorescence intensities for each fluorophore. However the methods ofthe invention are not restricted to rectangular boundaries. Anyboundary, closed or unclosed, can be employed. Furthermore, according tomethods of the invention, selections of droplet populations can be madeby any method, and is not restricted to threshold-based methods such asboundary selection.

FIG. 18A shows the droplet fluorescence intensities for the assay (righthistogram) when only one optical label was selected (left histogram).The lines overlaid on the histogram of the optical labels identify therectangular boundary used to select just the optical label with thelowest fluorescence for both fluorophores. Both histograms showed onlythe droplets that were selected. After selection, four distinct clustersof droplets appeared in the assay histogram, three for the differentassays (in this case, assays for SMN1, SMN2, and TERT, where TERT isanother common reference gene) and one for the empty droplets. The copynumbers for SMN1 and SMN2 were measured by the same methods of theinvention as described above for the 5-plex SMA assay, with values of1.8 and 0.94 close to the expected values of 2 and 1, respectively. Thesame assay was encoded with two other optical labels, and theirselections are shown in FIGS. 18B and C. Similar results were achieved,with an overall measurement of 1.9±0.1 and 0.9±0.1 copies of SMN1 andSMN2 respectively, showing the measurement to be accurate withinexperimental uncertainty.

FIGS. 19A, B, and C show optical label selections for a different assay(TERT, c.5C in the SMN1 gene, and BCKDHA (labeled E1a in the figure)).In each case four distinct clusters also appeared, and by the samemethods of the invention above, accurate measurements of gene copynumber were made for c.5C and BCKDHA, referenced to TERT, of 2.9±0.1 and2.0±0.2 compared to 3 and 2, respectively. FIGS. 20A, B, and C showoptical label selections for a third assay (TERT, c.88G in the SMN1gene, and RNaseP, where RNaseP is a common reference gene). Accurategene copy numbers of 2.1±0.1 were measured for both c.88G and RNaseP,referenced to TERT, compared to the expected value of 2.

In summary, the demonstration here shows use of nine different opticallabels to enable independent measurement of three triplex assays in asingle experiment. Although some of the optical labels encoded forredundant assays in this example (there were only three different assaysdespite having nine optical labels), the invention is not constrained toany particular formatting of assays and optical labels. Embodiments ofthe invention include formats where all of the assays are the sameacross all of the optical labels; where none of the assays are the sameacross all of the optical labels; where some of the assays are the sameacross all of the optical labels; where some of the assays have greaterplexity than others across all of the optical labels; where all of theassays have the same plexity across all of the optical labels; and anyother arrangements of assays across all of the optical labels areconsidered.

Although two different fluorophores were used to create the opticallabels in this example, the invention is not constrained to anyparticular number of fluorophores comprising the optical labels.Embodiments of the invention include optical labels comprised of 1fluorophore, or 2 fluorophores, or 3 fluorophores, or 4 fluorophores, orup to 10 fluorophores, or up to 20 fluorophores. Optical labels can alsocomprise more than 20 fluorophores.

Although solely triplex assays were used in the example demonstrationhere, the invention is not constrained to use of triplex assays withoptical labels. Embodiments of the invention include plexities of thefollowing amounts when used with optical labels: single plex, duplex,triplex, 4-plex, up to 10-plex, up to 20-plex, up to 50-plex, and up to100-plex. Embodiments of the invention also include plexities exceeding100 when used with optical labels.

Another method of the invention involves the use of droplet merging,instead of co-flow, for combining multiplexing with optical labels. Ademonstration using droplet merging was performed with the same 3×3×3assay as in the preceding example with co-flow. The assays (probes andprimers) combined with their unique optical labels were firstencapsulated into droplets along with the PCR master mix. Subsequently,according to methods of the invention described above, a librarycontaining a mixture of droplets from all nine optically labeled assayswas merged one-to-one with droplets containing template DNA from thesame patient as in the preceding example. As another method of theinvention, the droplet merge was performed using a lambda-injector stylemerge module, as described in U.S. Provisional Application Ser. No.61/441,985, incorporated by reference herein. Aside from the differencesbetween co-flow and merge, the assays and experimental procedures wereidentical to those above for the co-flow experiment. FIG. 21 shows 2-Dhistograms of droplet fluorescence intensity for the optical labels andthe assays that are similar to those in FIGS. 17-20. As in the case forco-flow, upon selection of droplets containing individual opticallabels, the expected distinct clusters of droplets corresponding to eachassay were clearly evident. Furthermore for each assay the measured genecopy number matched or very nearly matched the expected values withinexperimental uncertainty (See Table 1).

TABLE 1 Gene copy number measurements from the 3 × 3 × 3 assay. Gene orMeasured Expected genotype copy number copy number SMN1 1.98 ± 0.09 2SMN2 0.99 ± 0.04 1 c.5C in SMN1 3.01 ± 0.06 3 c.88G in SMN1 2.15 ± 0.082 BCKDHA 2.00 ± 0.05 2 RNaseP 2.11 ± 0.16 2

Although methods of the invention include using either microfluidicswith co-flow or droplet merging, the invention is not limited in thisregard. Any fluidic method capable of generating optically labeleddroplets that also contain fluorogenic DNA hybridization probes areconsidered. For example, other embodiments well known in the art aremixing optical labels and assays in the macrofluidic environment beforeinjection into a droplet generating chip; and mixing optical labels andassays thoroughly upstream from the droplet forming module in dedicatedmixing modules, such as with a serpentine mixer.

Data Analysis

One method of the invention involves histogram-based data presentationand analysis for identifying and characterizing populations ofstatistically similar droplets that arise from unique probe signatures(color and intensity), and for discriminating one population of dropletsfrom the others. Another method of the invention involveshistogram-based data presentation and analysis for identifying andselecting populations of droplets based on unique signatures fromoptical labels. Examples of one and two-dimensional histograms have beenprovided for these methods, but the invention is not limited in thisregard. As described above, it is anticipated that greater numbers ofcolors will be used for both multiplexing and for optical labels. Hence,embodiments of the invention include histograms of dimensionalitygreater than two, such as 3, or 4, or up to 10, or up to 20. Histogramsof dimensionality greater than 20 are also incorporated into theinvention.

Another method of the invention involves the selection of dropletswithin histograms, either for counting, or for assay selection as in theuse of optical labels, or for any other purpose. Methods of theinvention include selections by boundaries, either closed or unclosed,of any possible shape and dimension. Methods of the invention alsoinclude selections of droplets that exhibit fluorescence from singletypes of fluorophores, or from multiple types of fluorophores, such asarising from multiple probes against a common DNA target.

Polymerase Error Correction

For applications requiring very high sensitivity, such as searching forrare mutations amidst an abundance of wild-type DNA, false positiveresults can arise from errors from the DNA polymerase itself. Forexample, during one of the early thermal cycles the polymerase mightsynthesize the mutant strand of DNA from a wild-type template. This typeof error is most likely to occur when the difference between the mutantand the wild-type is very small, such as single nucleotide polymorphism(SNP). In this method of the invention, each droplet contains only asingle target nucleic acid, if any at all. In the preferred embodiment,this is accomplished under the conditions of terminal dilution. Dropletsthat contain amplification products that are a wild-type of the targetare detected based on emission from the fluorophore that is releasedfrom the probe that hybridizes to the wild-type of the target. Dropletsthat contain the variant of the target are detected based on emissionfrom the fluorophore that is released from the probe that hybridizes tothe variant of the target. Since each droplet starts with only a singlenucleic acid molecule, the resultant amplification products in eachdroplet are either homogeneous for the target or homogenous for thevariant of the target.

However, certain droplets will contain a heterogeneous mixture of bothtarget and target variant due to polymerase errors during the PCRreaction. Error rates in PCR vary according to the precise nucleic acidsequence, the thermostable enzyme used, and the in vitro conditions ofDNA synthesis. For example, the error frequency (mutations pernucleotide per cycle) during PCR catalyzed by the thermostable Thermusaquaticus (Taq) DNA polymerase vary more than 10-fold, from −2×10⁻⁴ to<1×10⁻⁵. Eckert et al. (Genome Res. 1:17-24, 1991), the content of whichis incorporated by reference herein in its entirety. Polymerase-mediatederrors at a frequency of 1 mutation per 10,000 nucleotides per cycle arean important consideration for any PCR application that begins with asmall amount of starting material (e.g., less than a total of 10,000nucleotides of target DNA) or that focuses on individual DNA moleculesin the final PCR population.

The proportion of DNA molecules that contain sequence changes is afunction of the error rate per nucleotide per cycle, the number ofamplification cycles and the starting population size. The population ofaltered DNA molecules arises during PCR from two sources: (1) new errorsat each PCR cycle; and (2) amplification of DNA molecules containingerrors from previous cycles. The formula f=np/2 describes the averagemutation frequency (f) for PCR amplification as a function of thepolymerase error rate per nucleotide per cycle (p) and the number ofcycles (n), assuming that p is constant at each cycle. Due to theexponential nature of PCR, the occurrence of an early error can increasethe final error frequency above the average described by f=np/2, becausethe variant DNA molecule will be amplified with each cycle, resulting inpopulations with a larger than average number of variants.

A polymerase error that converts a wild-type of the target to a variantof the target during an early round of amplification results in aheterogeneous population of target and target variant in a droplet, andmay lead to a droplet being incorrectly identified as containing avariant of the target, i.e., a false positive. Such false positivesgreatly impact the validity and precision of digital PCR results.

Methods of the invention are able to detect which droplets contain aheterogeneous population of molecules and are able to exclude thosedroplets from analysis. As droplets containing amplified product flow ina channel through the detector module, the module is able to detect thefluorescent emission in each droplet. Droplets that produce only asingle signal are classified as droplets that contain a homogeneouspopulation of target. Since probes that hybridize to the wild-type ofthe target have a different fluorophore attached than probes thathybridize to a variant of the wild-type of the target, methods of theinvention can classify each droplet as containing either a homogeneouspopulation of amplicons of the target or a homogeneous population ofamplicons of the variant of the target.

Droplets that produce two signals are classified as droplets thatcontain a heterogeneous population of molecules. Since each dropletstarted with at most a single target nucleic acid, a droplet thatincludes amplification products that are both amplicons of the targetand amplicons of a variant of the target are droplets in which thevariant of the target was produced by a polymerase error during the PCRreaction, most likely a polymerase error during an early cycle of thePCR reaction. Such droplets are detected and excluded from analysis.

Analysis

Analyze is then performed on only the droplets that contain ahomogeneous population of molecules. The analysis may be based oncounting, i.e., determining a number of droplets that contain onlywild-type target, and determining a number of droplets that contain onlya variant of the target. Such methods are well known in the art. See,e.g., Lapidus et al. (U.S. Pat. Nos. 5,670,325 and 5,928,870) and Shuberet al. (U.S. Pat. Nos. 6,203,993 and 6,214,558), the content of each ofwhich is incorporated by reference herein in its entirety.

Generally, the presence of droplets containing only variant isindicative of a disease, such as cancer. In certain embodiments, thevariant is an allelic variant, such as an insertion, deletion,substitution, translocation, or single nucleotide polymorphism (SNP).

Biomarkers that are associated with cancer are known in the art.Biomarkers associated with development of breast cancer are shown inErlander et al. (U.S. Pat. No. 7,504,214), Dai et al. (U.S. Pat. Nos.7,514,209 and 7,171,311), Baker et al. (U.S. Pat. No. 7,056,674 and U.S.Pat. No. 7,081,340), Erlander et al. (US 2009/0092973). The contents ofthe patent application and each of these patents are incorporated byreference herein in their entirety. Biomarkers associated withdevelopment of cervical cancer are shown in Patel (U.S. Pat. No.7,300,765), Pardee et al. (U.S. Pat. No. 7,153,700), Kim (U.S. Pat. No.6,905,844), Roberts et al. (U.S. Pat. No. 6,316,208), Schlegel (US2008/0113340), Kwok et al. (US 2008/0044828), Fisher et al. (US2005/0260566), Sastry et al. (US 2005/0048467), Lai (US 2008/0311570)and Van Der Zee et al. (US 2009/0023137). Biomarkers associated withdevelopment of vaginal cancer are shown in Giordano (U.S. Pat. No.5,840,506), Kruk (US 2008/0009005), Hellman et al. (Br J. Cancer.100(8):1303-1314, 2009). Biomarkers associated with development of braincancers (e.g., glioma, cerebellum, medulloblastoma, astrocytoma,ependymoma, glioblastoma) are shown in D'Andrea (US 2009/0081237),Murphy et al. (US 2006/0269558), Gibson et al. (US 2006/0281089), andZetter et al. (US 2006/0160762). Biomarkers associated with developmentof renal cancer are shown in Patel (U.S. Pat. No. 7,300,765), Soyupak etal. (U.S. Pat. No. 7,482,129), Sahin et al. (U.S. Pat. No. 7,527,933),Price et al. (U.S. Pat. No. 7,229,770), Raitano (U.S. Pat. No.7,507,541), and Becker et al. (US 2007/0292869). Biomarkers associatedwith development of hepatic cancers (e.g., hepatocellular carcinoma) areshown in Horne et al. (U.S. Pat. No. 6,974,667), Yuan et al. (U.S. Pat.No. 6,897,018), Hanausek-Walaszek et al. (U.S. Pat. No. 5,310,653), andLiew et al. (US 2005/0152908). Biomarkers associated with development ofgastric, gastrointestinal, and/or esophageal cancers are shown in Changet al. (U.S. Pat. No. 7,507,532), Bae et al. (U.S. Pat. No. 7,368,255),Muramatsu et al. (U.S. Pat. No. 7,090,983), Sahin et al. (U.S. Pat. No.7,527,933), Chow et al. (US 2008/0138806), Waldman et al. (US2005/0100895), Goldenring (US 2008/0057514), An et al. (US2007/0259368), Guilford et al. (US 2007/0184439), Wirtz et al. (US2004/0018525), Filella et al. (Acta Oncol. 33(7):747-751, 1994), Waldmanet al. (U.S. Pat. No. 6,767,704), and Lipkin et al. (Cancer Research,48:235-245, 1988). Biomarkers associated with development of ovariancancer are shown in Podust et al. (U.S. Pat. No. 7,510,842), Wang (U.S.Pat. No. 7,348,142), O'Brien et al. (U.S. Pat. Nos. 7,291,462,6,942,978, 6,316,213, 6,294,344, and 6,268,165), Ganetta (U.S. Pat. No.7,078,180), Malinowski et al. (US 2009/0087849), Beyer et al. (US2009/0081685), Fischer et al. (US 2009/0075307), Mansfield et al. (US2009/0004687), Livingston et al. (US 2008/0286199), Farias-Eisner et al.(US 2008/0038754), Ahmed et al. (US 2007/0053896), Giordano (U.S. Pat.No. 5,840,506), and Tchagang et al. (Mol Cancer Ther, 7:27-37, 2008).Biomarkers associated with development of head-and-neck and thyroidcancers are shown in Sidransky et al. (U.S. Pat. No. 7,378,233),Skolnick et al. (U.S. Pat. No. 5,989,815), Budiman et al. (US2009/0075265), Hasina et al. (Cancer Research, 63:555-559, 2003),Kebebew et al. (US 2008/0280302), and Ralhan (Mol Cell Proteomics,7(6):1162-1173, 2008). The contents of each of the articles, patents,and patent applications are incorporated by reference herein in theirentirety. Biomarkers associated with development of colorectal cancersare shown in Raitano et al. (U.S. Pat. No. 7,507,541), Reinhard et al.(U.S. Pat. No. 7,501,244), Waldman et al. (U.S. Pat. No. 7,479,376);Schleyer et al. (U.S. Pat. No. 7,198,899); Reed (U.S. Pat. No.7,163,801), Robbins et al. (U.S. Pat. No. 7,022,472), Mack et al. (U.S.Pat. No. 6,682,890), Tabiti et al. (U.S. Pat. No. 5,888,746), Budiman etal. (US 2009/0098542), Karl (US 2009/0075311), Arjol et al. (US2008/0286801), Lee et al. (US 2008/0206756), Mori et al. (US2008/0081333), Wang et al. (US 2008/0058432), Belacel et al. (US2008/0050723), Stedronsky et al. (US 2008/0020940), An et al. (US2006/0234254), Eveleigh et al. (US 2004/0146921), and Yeatman et al. (US2006/0195269). Biomarkers associated with development of prostate cancerare shown in Sidransky (U.S. Pat. No. 7,524,633), Platica (U.S. Pat. No.7,510,707), Salceda et al. (U.S. Pat. No. 7,432,064 and U.S. Pat. No.7,364,862), Siegler et al. (U.S. Pat. No. 7,361,474), Wang (U.S. Pat.No. 7,348,142), Ali et al. (U.S. Pat. No. 7,326,529), Price et al. (U.S.Pat. No. 7,229,770), O'Brien et al. (U.S. Pat. No. 7,291,462), Golub etal. (U.S. Pat. No. 6,949,342), Ogden et al. (U.S. Pat. No. 6,841,350),An et al. (U.S. Pat. No. 6,171,796), Bergan et al. (US 2009/0124569),Bhowmick (US 2009/0017463), Srivastava et al. (US 2008/0269157),Chinnaiyan et al. (US 2008/0222741), Thaxton et al. (US 2008/0181850),Dahary et al. (US 2008/0014590), Diamandis et al. (US 2006/0269971),Rubin et al. (US 2006/0234259), Einstein et al. (US 2006/0115821), Pariset al. (US 2006/0110759), Condon-Cardo (US 2004/0053247), and Ritchie etal. (US 2009/0127454). Biomarkers associated with development ofpancreatic cancer are shown in Sahin et al. (U.S. Pat. No. 7,527,933),Rataino et al. (U.S. Pat. No. 7,507,541), Schleyer et al. (U.S. Pat. No.7,476,506), Domon et al. (U.S. Pat. No. 7,473,531), McCaffey et al.(U.S. Pat. No. 7,358,231), Price et al. (U.S. Pat. No. 7,229,770), Chanet al. (US 2005/0095611), Mitchl et al. (US 2006/0258841), and Faca etal. (PLoS Med 5(6):e123, 2008). Biomarkers associated with developmentof lung cancer are shown in Sahin et al. (U.S. Pat. No. 7,527,933),Hutteman (U.S. Pat. No. 7,473,530), Bae et al. (U.S. Pat. No.7,368,255), Wang (U.S. Pat. No. 7,348,142), Nacht et al. (U.S. Pat. No.7,332,590), Gure et al. (U.S. Pat. No. 7,314,721), Patel (U.S. Pat. No.7,300,765), Price et al. (U.S. Pat. No. 7,229,770), O'Brien et al. (U.S.Pat. No. 7,291,462 and U.S. Pat. No. 6,316,213), Muramatsu et al. (U.S.Pat. No. 7,090,983), Carson et al. (U.S. Pat. No. 6,576,420), Giordano(U.S. Pat. No. 5,840,506), Guo (US 2009/0062144), Tsao et al. (US2008/0176236), Nakamura et al. (US 2008/0050378), Raponi et al. (US2006/0252057), Yip et al. (US 2006/0223127), Pollock et al. (US2006/0046257), Moon et al. (US 2003/0224509), and Budiman et al. (US2009/0098543). Biomarkers associated with development of skin cancer(e.g., basal cell carcinoma, squamous cell carcinoma, and melanoma) areshown in Roberts et al. (U.S. Pat. No. 6,316,208), Polsky (U.S. Pat. No.7,442,507), Price et al. (U.S. Pat. No. 7,229,770), Genetta (U.S. Pat.No. 7,078,180), Carson et al. (U.S. Pat. No. 6,576,420), Moses et al.(US 2008/0286811), Moses et al. (US 2008/0268473), Dooley et al. (US2003/0232356), Chang et al. (US 2008/0274908), Alani et al. (US2008/0118462), Wang (US 2007/0154889), and Zetter et al. (US2008/0064047). Biomarkers associated with development of multiplemyeloma are shown in Coignet (U.S. Pat. No. 7,449,303), Shaughnessy etal. (U.S. Pat. No. 7,308,364), Seshi (U.S. Pat. No. 7,049,072), andShaughnessy et al. (US 2008/0293578, US 2008/0234139, and US2008/0234138). Biomarkers associated with development of leukemia areshown in Ando et al. (U.S. Pat. No. 7,479,371), Coignet (U.S. Pat. No.7,479,370 and U.S. Pat. No. 7,449,303), Davi et al. (U.S. Pat. No.7,416,851), Chiorazzi (U.S. Pat. No. 7,316,906), Seshi (U.S. Pat. No.7,049,072), Van Baren et al. (U.S. Pat. No. 6,130,052), Taniguchi (U.S.Pat. No. 5,643,729), Insel et al. (US 2009/0131353), and Van Bockstaeleet al. (Blood Rev. 23(1):25-47, 2009). Biomarkers associated withdevelopment of lymphoma are shown in Ando et al. (U.S. Pat. No.7,479,371), Levy et al. (U.S. Pat. No. 7,332,280), and Arnold (U.S. Pat.No. 5,858,655). Biomarkers associated with development of bladder cancerare shown in Price et al. (U.S. Pat. No. 7,229,770), Orntoft (U.S. Pat.No. 6,936,417), Haak-Frendscho et al. (U.S. Pat. No. 6,008,003),Feinstein et al. (U.S. Pat. No. 6,998,232), Elting et al. (US2008/0311604), and Wewer et al. (2009/0029372). The content of each ofthe above references is incorporated by reference herein in itsentirety.

In certain embodiments, methods of the invention may be used to monitora patient for recurrence of a cancer. Since the patient has already beentreated for the cancer, the genetic profile and particular mutation(s)associated with that patient's cancer are already known. Probes may bedesigned that specifically hybridize to the region of the nucleic acidthat contains the mutation(s) that is indicative of the cancer for whichthe patient was previously treated. A patient's sample (e.g., pus,sputum, semen, urine, blood, saliva, stool, or cerebrospinal fluid) maythen be analyzed as described above to determine whether the mutantallele(s) is detected in the sample, the presence of which beingindicative of recurrence of the cancer.

Droplet Sorting

Methods of the invention may further include sorting the droplets basedupon whether the droplets contain a homogeneous population of moleculesor a heterogeneous population of molecules. A sorting module may be ajunction of a channel where the flow of droplets can change direction toenter one or more other channels, e.g., a branch channel, depending on asignal received in connection with a droplet interrogation in thedetection module. Typically, a sorting module is monitored and/or underthe control of the detection module, and therefore a sorting module maycorrespond to the detection module. The sorting region is incommunication with and is influenced by one or more sorting apparatuses.

A sorting apparatus includes techniques or control systems, e.g.,dielectric, electric, electro-osmotic, (micro-) valve, etc. A controlsystem can employ a variety of sorting techniques to change or directthe flow of molecules, cells, small molecules or particles into apredetermined branch channel. A branch channel is a channel that is incommunication with a sorting region and a main channel. The main channelcan communicate with two or more branch channels at the sorting moduleor branch point, forming, for example, a T-shape or a Y-shape. Othershapes and channel geometries may be used as desired. Typically, abranch channel receives droplets of interest as detected by thedetection module and sorted at the sorting module. A branch channel canhave an outlet module and/or terminate with a well or reservoir to allowcollection or disposal (collection module or waste module, respectively)of the molecules, cells, small molecules or particles. Alternatively, abranch channel may be in communication with other channels to permitadditional sorting.

A characteristic of a fluidic droplet may be sensed and/or determined insome fashion, for example, as described herein (e.g., fluorescence ofthe fluidic droplet may be determined), and, in response, an electricfield may be applied or removed from the fluidic droplet to direct thefluidic droplet to a particular region (e.g. a channel). In certainembodiments, a fluidic droplet is sorted or steered by inducing a dipolein the uncharged fluidic droplet (which may be initially charged oruncharged), and sorting or steering the droplet using an appliedelectric field. The electric field may be an AC field, a DC field, etc.For example, a channel containing fluidic droplets and carrier fluid,divides into first and second channels at a branch point. Generally, thefluidic droplet is uncharged. After the branch point, a first electrodeis positioned near the first channel, and a second electrode ispositioned near the second channel. A third electrode is positioned nearthe branch point of the first and second channels. A dipole is theninduced in the fluidic droplet using a combination of the electrodes.The combination of electrodes used determines which channel will receivethe flowing droplet. Thus, by applying the proper electric field, thedroplets can be directed to either the first or second channel asdesired. Further description of droplet sorting is shown for example inLink et al. (U.S. patent application numbers 2008/0014589, 2008/0003142,and 2010/0137163) and European publication number EP2047910 to RaindanceTechnologies Inc.

Based upon the detected signal at the detection module, dropletscontaining a heterogeneous population of molecules are sorted away fromdroplets that contain a homogeneous population of molecules. Dropletsmay be further sorted to separate droplets that contain a homogeneouspopulation of amplicons of the target from droplets that contain ahomogeneous population of amplicons of the variant of the target.

Release of Target from Droplet

Methods of the invention may further involve releasing amplified targetmolecules from the droplets for further analysis. Methods of releasingamplified target molecules from the droplets are shown in for example inLink et al. (U.S. patent application numbers 2008/0014589, 2008/0003142,and 2010/0137163) and European publication number EP2047910 to RainDanceTechnologies Inc.

In certain embodiments, sample droplets are allowed to cream to the topof the carrier fluid. By way of non-limiting example, the carrier fluidcan include a perfluorocarbon oil that can have one or more stabilizingsurfactants. The droplet rises to the top or separates from the carrierfluid by virtue of the density of the carrier fluid being greater thanthat of the aqueous phase that makes up the droplet. For example, theperfluorocarbon oil used in one embodiment of the methods of theinvention is 1.8, compared to the density of the aqueous phase of thedroplet, which is 1.0.

The creamed liquids are then placed onto a second carrier fluid whichcontains a de-stabilizing surfactant, such as a perfluorinated alcohol(e.g. 1H,1H,2H,2H-Perfluoro-1-octanol). The second carrier fluid canalso be a perfluorocarbon oil. Upon mixing, the aqueous droplets beginsto coalesce, and coalescence is completed by brief centrifugation at lowspeed (e.g., 1 minute at 2000 rpm in a microcentrifuge). The coalescedaqueous phase can now be removed and the further analyzed.

The released amplified material can also be subjected to furtheramplification by the use tailed primers and secondary PCR primers. Inthis embodiment the primers in the droplet contain an additionalsequence or tail added onto the 5′ end of the sequence specific portionof the primer. The sequences for the tailed regions are the same foreach primer pair and are incorporated onto the 5′ portion of theamplicons during PCR cycling. Once the amplicons are removed from thedroplets, another set of PCR primers that can hybridize to the tailregions of the amplicons can be used to amplify the products throughadditional rounds of PCR. The secondary primers can exactly match thetailed region in length and sequence or can themselves containadditional sequence at the 5′ ends of the tail portion of the primer.During the secondary PCR cycling these additional regions also becomeincorporate into the amplicons. These additional sequences can include,but are not limited to adaptor regions utilized by sequencing platformsfor library preparation and sequencing, sequences used as a barcodingfunction for the identification of samples multiplexed into the samereaction. molecules for the separation of amplicons from the rest of thereaction materials such as biotin, digoxin, peptides, or antibodies andmolecules such as fluorescent markers that can be used to identify thefragments.

In certain embodiments, the amplified target molecules are sequenced. Ina particular embodiment, the sequencing is single-moleculesequencing-by-synthesis. Single-molecule sequencing is shown for examplein Lapidus et al. (U.S. Pat. No. 7,169,560), Quake et al. (U.S. Pat. No.6,818,395), Harris (U.S. Pat. No. 7,282,337), Quake et al. (U.S. patentapplication number 2002/0164629), and Braslaysky, et al., PNAS (USA),100: 3960-3964 (2003), the contents of each of these references isincorporated by reference herein in its entirety.

Briefly, a single-stranded nucleic acid (e.g., DNA or cDNA) ishybridized to oligonucleotides attached to a surface of a flow cell. Thesingle-stranded nucleic acids may be captured by methods known in theart, such as those shown in Lapidus (U.S. Pat. No. 7,666,593). Theoligonucleotides may be covalently attached to the surface or variousattachments other than covalent linking as known to those of ordinaryskill in the art may be employed. Moreover, the attachment may beindirect, e.g., via the polymerases of the invention directly orindirectly attached to the surface. The surface may be planar orotherwise, and/or may be porous or non-porous, or any other type ofsurface known to those of ordinary skill to be suitable for attachment.The nucleic acid is then sequenced by imaging the polymerase-mediatedaddition of fluorescently-labeled nucleotides incorporated into thegrowing strand surface oligonucleotide, at single molecule resolution.

Experimental Detail

What follows is experimental detail for the various experiments detailsabove.

Primers and Probes

All TaqMan® primers and probes used here are listed in Table 2. Unlessotherwise noted by reference in the table, the primers and probes weredesigned with the “Custom TaqMan® Assay Design Tool” from AppliedBiosystems Inc. (ABI) and procured through ABI (Carlsbad, Calif.).Probes were labeled with 6-carboxyfluorescein (FAM, λex 494 nm \λem 494nm) or VIC™ (from ABI, λex 538 nm \λem 554 nm).

TABLE 2 5-plex Primers assay Target Assay (5′ to 3′) Probe (5′ to 3′)conditions Ref SMN1 Copy  (f) AATGCTTTTTAA- FAM- 0.37x Anhuf numberCATCCATATAAAGCT CAGGGTTTC*AGACAAA- et al.,  (SEQ ID NO.: 1) MGBNFQ  2003(SEQ ID NO.: 3) (r) CCTTAATTTAAG- GAATGTGAGCACC (SEQ ID NO.: 2) SMN2Copy  (f) AATGCTTTTTAA- FAM-TGATTTTGTCTA*AAA- 0.76x Anhuf numberCATCCATATAAAGCT CCC-MGBNFQ  et al.,  (SEQ ID NO.: 4) (SEQ ID NO.: 6)2003 (r) CCTTAATTTAAG- GAATGTGAGCACC (SEQ ID NO.: 5) BCKDHA Copy (f) CAACCTACTCTT- (FAM/VIC)- FAM: 0.18x DiMatteo number CTCAGACGTGTA CAGGAGATGCCCG- VIC: 0.56x et al.,  (SEQ ID NO.: 7) CCCAGCTC-TAMRA  2008(SEQ ID NO.: 9) (r) TCGAAGTGATCC- AGTGGGTAGTG  (SEQ ID NO.: 8) c.815A >G SNP (f) TGCTGATGCTTT- (A) (FAM/VIC)- 0.9x GGGAAGTATGTTA  CATGAGTGG- (SEQ ID NO.: 10) CTA*TCATAC-MGBNFQ (SEQ ID NO.: 11) (r) TGTCAGGAAAAG-(G) FAM- FAM: 0.9x ATGCTGAGTGATT  ATGAGTGGCTG*TC-  VIC: 0.45x(SEQ ID NO.: 12) ATAC-MGBNFQ (SEQ ID NO.: 13);  VIC-CATGA-GTGGCTG*TCATAC- MGBNFQ (SEQ ID NO.: 14) RNaseP Copy  Unknown unknown n/aStandard number product, 4403326, ABI 5′-exonuclease genotyping assaydesign. Assay conditions in column 5 are specific to the multiplexed SMAassay. References: D. Anhuf, T. Eggermann, S. Rudnik-Schöneborn and K.Zerres, Hum Mutat., 2003, 22, 74-78; D. DiMatteo, S. Callahan and E. B.Kmiec, Exp Cell Res., 2008, 15, 878-886.

Target DNA

For some genetic targets, BCKDHA and SMN2, plasmid DNA was synthesized(GeneArt, Regensburg, Germany) containing the sequence spanning betweenthe primers (see Table 2) and cloned into the GeneArt standard vector(2.5 kb). The target fragment was released from the cloning vector byrestriction digestion with SfiI to avoid any DNA supercoiling that mightaffect the assay. For simplicity, these gene fragments are called“plasmid DNA” throughout the text. A string of different gene fragmentswas also synthesized (GeneArt) and cloned into the GeneArt standardvector for demonstration of multiplexed reactions, called an “artificialchromosome” in the text. In this case, the fragments were separated fromeach other by restriction digestion at flanking EcoRV sites. Human DNAwas obtained in already purified form from cell lines (See Table 3;Coriell, Camden, N.J.) and fragmented before use with a K7025-05nebulizer following manufacturer's instructions (Invitrogen, Carlsbad,Calif.). DNA concentration was quantified by measuring absorbance at 260nm on a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington,Del.).

TABLE 3 Map of patient numbers used in the text to Coriell cell lines.Patient Coriell cell number line 1 NA14638 2 NA14637 3 NA14097 4 NA140965 NA14094 6 NA14093 7 NA14092 8 NA14091 9 NA14090 10 NA13715 11 NA1371412 NA13712 13 NA13709 14 NA13707 15 NA13705 SMA carrier NA03814 SMA 1NA03813 SMA 2 NA00232 SMA 3 NA09677 SMA 4 NA10684

Microfluidics

Microfluidic chips were manufactured by conventional soft lithography.Molding masters were fabricated by spin coating SU-8 negativephotoresist (MicroChem. Corp., Newton, Mass.) onto 6 inch silicon wafersand transferring the fluidic features from photomasks (CAD/Art Services,Bandon, Oreg.) by contact lithography with an OAI Hybralign Series 200aligner (OAI, San Jose, Calif.). Chips contained channels with twodepths: deep channels with low hydrodynamic resistance (100±10 um) fortransporting fluid from external ports to the functional regions of thechip, and shallow channels (20±1 um) for droplet manipulation anddetection. SU-8 photoresists 2100 and 2025 were used for deep andshallow channels respectively. Polydimethylsiloxane (PDMS) (Sylgard®184, Dow Corning, Midland, Mich.) chips were molded from the negativemasters within mold housings of custom design. Glass cover slides werepermanently bonded to the fluidic side of the chips by surfaceactivation in an AutoGlow™ oxygen plasma system (Glow Research, Phoenix,Ariz.) followed by immediate contact bonding. To create hydrophobicsurfaces, the microfluidic channels were exposed for ^(˜)2 min to1H,1H,2H,2H-perfluorodecyltrichlorosilane (Alfa Aesar, Ward Hill, Mass.)dissolved in FC-3283 (3M Specialty Materials, St. Paul, Minn.) preparedas a mixture of 18 g silane in 100 uL solvent.

Two different microfluidic devices were used, one for droplet generationand the other for fluorescence readout after thermal cycling. Thedroplet generation chip created an emulsion of uniformly sized aqueousdroplets of template DNA and PCR master mix that were suspended in aninert fluorinated oil with an emulsion stabilizing surfactant, called“carrier oil” from this point forward (REB carrier oil; RainDanceTechnologies, Lexington, Mass.). Droplets were generated in across-shaped microfluidic intersection, or “nozzle”. As shown in FIG. 3a, under typical operation the aqueous phase flowed into the nozzle fromthe right (160 μL/hr), joining flows of the carrier oil from the top andbottom (750 uL/hr of total oil), and producing 4 pL droplets at a rateof 11 kHz. The channel widths at the intersection measured 15 um for theaqueous inlet, 12.5 for the oil inlets, and 15 um widening to 40 um atthe outlet. Flow was driven by custom OEM pumps (IDEX Corporation,Northbrook, Ill.).

Approximately 25 uL of the PCR reaction mixture was collected as anemulsion from the droplet generation chip and thermally cycled in a DNAEngine (Bio-Rad, Hercules, Calif.). The reaction mixture contained 1×TaqMan® universal PCR master mix (Applied Biosystems, Carlsbad, Calif.),0.2 mM dNTP (Takara Bio, Madison, Wis.), and various amounts of primerpairs and probes as described in the results. 1× assay concentration isdefined as 0.2 μM probes with 0.9 μM primers. In all cases, when variedfrom the 1× concentration, the primers and probes were varied by thesame amount. The cycler program included a 10 min hot start at 95° C.,and 45 cycles of 15 s at 95° C. and 60 s at 60° C.

The droplets became concentrated during off-chip handling because thecarrier oil is more dense than the aqueous phase and drained down fromthe emulsion. Hence the droplets were reinjected into the readout chipas a tightly packed emulsion that required dilution prior to readout toproperly distinguish one droplet from another. A “spacer” nozzle similarto the droplet generation nozzle above was used to inject uniform plugsof extra carrier oil between droplets immediately before readout. Asshown in FIG. 3 b, the droplet entrance into the nozzle tapered downinto a constriction about the size of an individual droplet forcing thedroplets to enter the nozzle in single file and consequently at a stablerate. Opposed flow of the carrier oil from the top and bottom channelsseparated the droplets uniformly. The channel leaving the spacer nozzleincreased in width along the direction of flow, and the droplets wereinterrogated by laser induced fluorescence at the location along thechannel where the width was smaller than or equal to the dropletdiameter (marked with an arrow in FIG. 3 b). The nozzle dimensions were15 um for the droplet entrance and exit, and 20 um for the oil lines.

Instrumentation

Fluorescence readout was performed by conventional epifluorescencemicroscopy with a custom microscope. A 20 mW, 488 nm laser source (Cyan;Picarro, Sunnyvale, Calif.) was expanded 2× and focused by the objectivelens (20×/0.45 NA; Nikon, Japan) onto the microfluidic channel. Two bandpass filters discriminated the fluorescence collected through theobjective lens: 512/25 nm and 529/28 nm for FAM and VIC fluorophoresrespectively (Semrock, Rochester, N.Y.). Fluorescence was detected bytwo H5784-20 photomultipliers (Hamamatsu, Japan) and was typicallyrecorded at a 200 kHz sampling rate with a USB-6259 data acquisitioncard (National Instruments, Austin, Tex.). The data traces were smoothedby a seven-point, second-order Savitzky-Golay algorithm beforesubsequent analysis. Concurrent with the fluorescence read out, thedroplets were imaged through the same objective lens with backsideillumination from an 850 nm LED (TSHG6200; Vishay Semiconductors,Shelton, Conn.), a short pass filter to separate the optical paths forfluorescence detection and imaging, and a Guppy CCD camera (AlliedVision Technologies, Newburyport, Mass.). Droplets were imaged withshort illumination pulses (5-20 us) to avoid image streaking.

Data Analysis

Data was analyzed with custom LabView software (National Instruments,Austin, Tex.) that interpreted droplet events as contiguous bursts offluorescence intensity above a threshold value. The signal-to-noiseratio was generally quite high and the signal levels were consistentfrom day to day, hence a fixed threshold value of 50 mV was usedpredominantly, otherwise the threshold was set by eye. The peakfluorescence intensity was recorded for each droplet event for both VICand FAM fluorophores. Some coalescence of droplets did occur duringthermal cycling, typically as isolated events between two intactdroplets forming “doublets.” Doublets and the rare larger coalescedevents were easily filtered from the data set on based on the durationof the fluorescence burst.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein.

1-23. (canceled)
 24. A method for detecting target sequences from asample, the method comprising: providing a plurality of partitions eachcomprising a nucleic acid molecule, reagents for an amplificationreaction, and a plurality of optically labeled probe types each specificfor a different target sequence; amplifying a target sequence from thenucleic acid molecule in the partitions; and detecting light emittedfrom an optically labeled probe type specific to the amplified targetsequence in the partitions.
 25. The method of claim 24, furthercomprising, identifying the amplified target sequence in the partitionsusing a combination of a wavelength and an intensity of the detectedlight.
 26. The method of claim 25, wherein the step of identifyingcomprises identifying one or more of the partitions that do not comprisean amplified target sequence.
 27. The method of claim 25, wherein thestep of identifying comprises identifying up to 9 amplified targetsequences in the partitions using a combination of a first wavelength, asecond wavelength, and the intensity of the detected light.
 28. Themethod of claim 25, wherein the step of identifying comprises expressionanalysis of the amplified target sequence.
 29. The method of claim 25,wherein the nucleic acid molecules are from a sample, a first opticallylabeled probe type is specific for a reference target sequence, a secondoptically labeled probe type is specific for a copy number varianttarget sequence; and the step of identifying comprises determining anumber of the identified partitions comprising the reference targetsequence and a number of the identified partitions comprising the copynumber variant target sequence, wherein a ratio of the number ofreference partitions to the number of copy number variant partitionsdetermines a gene copy number of the copy number variant sequence. 30.The method of claim 29, wherein the sample is from a patient and thecopy number variant sequence comprises SMN1 or SMN2.
 31. The method ofclaim 24, wherein the optically labeled probe types each comprise aspecies of fluorescent label.
 32. The method of claim 31, wherein theoptically labeled probe types comprise hydrolysis probes and the speciesof fluorescent label comprises a VIC dye species or a FAM dye species.33. The method of claim 24, wherein the sample is a human tissue or bodyfluid.
 34. The method of claim 24, wherein a plurality of the dropletseach comprise no more than a single nucleic acid molecule.
 35. Themethod of claim 24, wherein the reagents for amplification comprise aplurality of different primer types each specific to amplify a differenttarget sequence.
 36. The method of claim 24, wherein at least one of theoptically labeled probe types specific for a target sequence comprises afirst sub-type comprising a first fluorescent label and a secondsub-type comprising a second fluorescent label different than the first,wherein the first and second sub-types are specific to the same targetsequence.
 37. The method of claim 24, wherein at least one of theoptically labeled probe types specific for a target sequence comprises afirst sub-type comprising a first fluorescent label and a secondsub-type without a fluorescent label, wherein the first and secondsub-types are specific to the same target sequence.
 38. The method ofclaim 24, wherein the partitions comprise droplets.
 39. A method fordetecting target sequences from a sample, the method comprising:providing a plurality of droplets each comprising reagents for anamplification reaction, and a plurality of different optically labeledprobe types each specific for a different target sequence; introducing anucleic acid molecule into a plurality of the droplets; amplifying atarget sequence from the nucleic acid molecule in the droplets; anddetecting light emitted from an optically labeled probe type specific tothe amplified target sequence in the droplets.
 40. The method of claim39, wherein the step of introducing comprises fusing each of thedroplets comprising the reagents for an amplification reaction, and theplurality of different optically labeled probe types with a seconddroplet that comprises the nucleic acid molecule.
 41. The method ofclaim 39, wherein the step of introducing comprises integrating aportion of a fluid stream comprising the nucleic acid molecule with eachof the droplets.
 42. The method of claim 39, further comprising,identifying the amplified target sequence in droplets using acombination of a wavelength and an intensity of the detected light. 43.The method of claim 42, wherein the step of identifying comprisesidentifying one or more of the droplets that do not comprise anamplified target sequence.
 44. The method of claim 42, wherein the stepof identifying comprises identifying up to 9 amplified target sequencesin the droplets using a combination of a first wavelength, a secondwavelength, and the intensity of the detected light.
 45. The method ofclaim 39, wherein the optically labeled probe types each comprise aspecies of fluorescent label.
 46. The method of claim 45, wherein theoptically labeled probe types comprise hydrolysis probes and the speciesof fluorescent label comprises a VIC dye species or a FAM dye species.47. The method of claim 39, wherein the reagents for amplificationcomprise a plurality of different primer types each specific to amplifya different target sequence.
 48. The method of claim 24, wherein thereagents for amplification comprise a polymerase species and a pluralityof deoxynucleotide species into the droplets.
 49. The method of claim24, wherein the step of introducing further comprises introducing apolymerase species and a plurality of deoxynucleotide species into thedroplets.
 50. The method of claim 39, wherein at least one of theoptically labeled probe types specific for a target sequence comprises afirst sub-type comprising a first fluorescent label and a secondsub-type comprising a second fluorescent label different than the first,wherein the first and second sub-types are specific to the same targetsequence.
 51. The method of claim 39, wherein at least one of theoptically labeled probe types specific for a target sequence comprises afirst sub-type comprising a first fluorescent label and a secondsub-type without a fluorescent label, wherein the first and secondsub-types are specific to the same target sequence.
 52. The method ofclaim 39, wherein the step of providing further comprises: providing oneor more optical identifier labels in the droplets and the reagents foramplification comprise a plurality of different primer types eachspecific to amplify a different target sequence, wherein each of theoptical identifier labels is specific to a different assay; and the stepof detecting further comprises detecting light emitted from the opticalidentifier labels.
 53. The method of claim 39, wherein the opticalidentifier labels comprise one or more fluorophores each of which isspectrally distinct from the optically labeled probe types.